Use of carbon monoxide dependent guanylyl cyclase modifiers to stimulate neuritogenesis

ABSTRACT

Disclosed herein are methods directed generally to the control of neural activity and for selectively and controllably inducing the in vivo genetic expression of one or more naturally occurring genetically encoded molecules in mammals. More particularly, the present invention selectively activates or derepresses genes encoding for specific naturally occurring molecules such as neurotrophic factors through the administration of carbon monoxide dependent guanylyl cyclase modulating purine derivatives. The methods of the present invention may be used to affect a variety of cellular and neurological activities and to therapeutically or prophylactically treat a wide variety of neurodegenerative, neurological, and cellular disorders.

CROSS-REFERENCES

This application is a continuation-in-part of copending application Ser.No. 09/086,878 filed May 29, 1998, now U.S. Pat. No. 6,027,936 entitled“Car Monoxide Dependent Guanylyl Cyclase Modifiers and Methods of Use,”which was a division of copending application Ser. No. 08/488,976, filedJun. 8, 1995, now U.S. Pat. No. 5,801,184, also entitled “CarbonMonoxide Dependent Guanylyl Cyclase Modifiers and Methods of Use,” whichwas a continuation-in-part of copending application Ser. No. 08/280,719filed Jul. 25, 1994, now U.S. Pat. No. 5,447,936, also entitled “CarbonMonoxide Dependent Guanylyl Cyclase Modifiers and Methods of Use.” Thedisclosures of these applications are hereby incorporated herein intheir entirety by this reference.

FIELD OF THE INVENTION

The present invention relates in general to the control of neuralactivity and to the treatment of neural disorders. More particularly,the present invention is directed to methods for the modification ofmammalian neural activity through the administration of carbon monoxidedependent guanylyl cyclase modulating purine derivatives whichselectively and controllably induce the in vivo genetic expression ofnaturally occurring genetically encoded molecules including neurotrophicfactors. The methods of the present invention may be used to affect avariety of neurological activities and to therapeutically orprophylactically treat a wide variety of neurodegenerative andneurological disorders.

BACKGROUND OF THE INVENTION

The evolution of the central nervous system in mammals was a naturalresponse to an increasingly complex environment requiring solutions todifficult problems. The resulting structure is an intricate biochemicalmatrix that is precisely controlled and attenuated to an elaboratesystem of chemically modulated regulatory pathways. Through an elaborateseries of highly specific chemical reactions, these pathways oversee anddirect every structural and operational aspect of the central nervoussystem and, through it, the organism itself. Normally, the complexinterplay of the various control systems cooperates to produce a highlyefficient, versatile central nervous system managed by the brain.Unfortunately, when the biochemical matrix of the central nervous systemis damaged, either through age, disease, or other reasons, the normalregulatory pathways may be incapable of effectively compensating for theloss. In such cases, it would be highly desirable to modify orsupplement the neural mechanisms to prevent such disorders or compensatefor them. That is the focus of the present invention.

More specifically, the mammalian brain is composed of approximately 10billion nerve cells or neurons surrounded by an even greater number ofsupport cells known as neuroglia or astrocyte cells. Neurons, like othercells of the body, are composed of a nucleus, a cytoplasm, and asurrounding cell membrane. However, unlike other cells, neurons alsopossess unique, fiber-like extensions allowing each individual nervecell to be networked with literally thousands of other nerve cells toestablish a neural infrastructure or network. Communication within thisintricate network provides the basis for all mental processes undertakenby an organism.

In each nerve cell, incoming signals are received by neural extensionsknown as dendrites which may number several thousand per nerve cell.Similarly, neural information is projected along nerve cell axons whichmay branch into as many as 10,000 different nerve endings. Together,these nerve cell axons and dendrites are generally termed neuritesthrough which each individual neuron can form a multitude of connectionswith other neurons. As a result, the number of possible neuralconnections in a healthy brain is in the trillions, giving rise totremendous mental capacity. Conversely, when the connections within theneural network break down as nerve cells die or degenerate due to age,disease, or direct physical insult, the mental capacity of the organismcan be severely compromised.

The connection of the individual axons with the dendrites or cell bodiesof other neurons takes place at junctions or sites known as synapses. Itis at the synapse that the individual neurons communicate with eachother through the flow of chemical messengers across the synapticjunction. The majority of these chemical messengers, orneurotransmitters, are small peptides, catecholamines, or amino acids.When the appropriate stimulus is received by a neural axon connection,the neurotransmitters diffuse across the synapse to the adjacent neuron,thereby conveying the stimulus to the next neuron across the neuralnetwork. Based on the complexity of the information transferred betweenthe nerve cells, it is currently believed that between 50 and 100distinct neurotransmitters are used to transmit signals in the mammalianbrain.

Quite recently, it was discovered that nitric oxide (NO) and carbonmonoxide (CO) may function as neurotransmitters. These gaseous moleculesappear to participate in a number of neuronal regulatory pathwaysaffecting cell growth and interactions. In the brain, as well as inother parts of the body, CO is produced by the enzyme heme oxygenase II(HO). Whether produced from the HO enzyme or from other sources, it isbelieved that when CO diffuses into a neuron it induces a rise in asecondary transmitter molecule known as cyclic guanosine monophosphate(cGMP) by modulating an enzyme known as guanylate cyclase or guanylylcyclase. Thus, CO acts as a signaling molecule in the guanylyl cyclaseregulatory pathway. The resultant increase in cGMP levels appears tomodify several neurotrophic factors as well as other neuronal factorswhich may induce, promote, or modify a variety of cellular functionsincluding cell growth and intercellular communication.

Neurotrophic factors are molecules that exert a variety of actionstimulating both the development and differentiation of neurons and themaintenance of cellular integrity and are required for the survival anddevelopment of neurons throughout the organism's life cycle. Generally,neurotrophic factors may be divided into two broad classes:neurotrophins and pleiotrophins. Pleiotrophins differ from theneurotrophins in that they lack a molecular signal sequencecharacteristic of molecules that are secreted from cells and in thatthey also affect many types of cells including neurons. Two effects ofneurotrophic factors are particularly important: (i) the prevention ofneuronal death and (ii) the stimulation of the outgrowth of neurites(either nascent axons or dendrites). In addition, it appears thatCO-induced neurotrophic factors may reduce the membrane potential ofnerve cells making it easier for the neurons to receive and transmitsignals.

Many of today's researchers believe that memory is associated with themodification of synaptic activity, wherein the synaptic connectionsbetween particular groups of brain neurons become strengthened orfacilitated after repeated activation. As a result, these modifiedconnections activate much more easily. This type of facilitation isbelieved to occur throughout the brain but may be particularly prominentin the hippocampus, a brain region which is critical for memory. Thestimulation of neuronal pathways within the hippocampus can produceenhanced synaptic transmission through these pathways for many daysfollowing the original stimulation. This process is known as long termpotentiation.

More particularly, long term potentiation is a form ofactivity-dependent synaptic electrical activity that is exhibited bymany neuronal pathways. In this state, generally accepted as a type ofcellular memory, nerve cells are more responsive to stimulation.Accordingly, it is widely believed that LTP provides an excellent modelfor understanding the cellular and molecular basis of synapticplasticity of the type that underlies learning and memory invertebrates, including man.

NO and CO are currently the leading candidates for messenger substancesthat facilitate LTP because inhibitors of these compounds retard theinduction of potentiation. The ability to modify neural activity and toincrease the ease of LTP using these or other signal transducers couldpotentially increase learning rates and cognitive powers, possibly,compensating for decreased mental acuity. Prior to the presentinvention, there were no known agents which could operate on thecellular level in vivo to reliably modify neural regulatory pathways soas to facilitate the LTP of neurons.

In contrast to the enhanced mental capacity provided by long-termpotentiation, mental functions may be impeded to varying degrees whenthe neuronal network is disrupted through the death or dysfunction ofconstituent nerve cells. While the decline in mental abilities isdirectly related to the disruption of the neural network, it isimportant to remember that the disruption is occurring on an individualcellular level. At this level the deleterious effects associated withneuronal disruption may be brought about by any one of a number offactors including neurodegenerative diseases and disorders, aging,trauma, and exposure to harmful chemical or environmental agents. Amongthe known neurological diseases which adversely impact neuronal functionare Alzheimer's disease and related disorders, Parkinson's disease,motor neuropathic diseases such as amyotrophic lateral sclerosis (LouGehrig's disease), cerebral palsy, multiple sclerosis, and Huntington'sdisease. Similar problems may be brought about by loss of neuronalconnectivity due to normal aging and to damage to neurons from stroke orother circulatory complications. Direct physical trauma or environmentalfactors including chemical agents, heavy metals, and the like may alsoprovoke neuronal dysfunction.

Whatever the cause of the neural disorder or dysfunction, the generalinability of damaged nerve cells to undergo substantial regrowth orregeneration under natural conditions has led to the proposal thatneurotrophic factors be administered to nerve cells in order to helprestore neuronal function by stimulating nerve growth and functions.Similarly, stimulating neuritogenesis, or the growth of neurites, byadministering neurotrophic factors may contribute to the ability ofsurviving neurons to form collateral connections and thereby restoreneural function.

At present, prior art techniques and compounds have not been effectiveor practical to directly administer neurotrophic factors to a patientsuffering from a neural disorder. In part, this is due to the complexmolecular interaction of the neurotrophic factors themselves and to thesynergistic regulation of neural cell growth and neuritogenesis.Neurotrophic factors are the result of a long chemical cascade which isexquisitely regulated on the molecular level by an intricate series oftransmitters and receptors. Accordingly, neuronal cells are influencedby a concert of different neurotrophic factors, each contributing todifferent aspects of neuronal development at different times.Neurotrophic factors are, effectively, the tail end of this cascade andthus are one of the most complex components of the regulatory pathway.As such, it was naive for prior art practitioners to assume that theunattenuated administration of single neurotrophic factors at randomtimes (from the cells' viewpoint) could substantially improve cellactivity or regeneration. In contrast, modification of the regulatorypathway earlier in the cascade could allow the proper growth factors tobe produced in the correct relative amounts and introduced into thecomplex cellular environment at the appropriate time.

Other practical considerations also preclude the prior art use ofneurotrophic factors to stimulate the regeneration of the neuronalnetwork. Neurotrophic factors (including neurotrophins andpleiotrophins) are large proteins and, as such, are not amenable tonormal routes of medical administration. For example, these proteinscannot be delivered to a patient or subject orally as the patient'sdigestive system would digest them before they reached their targetneural site. Moreover, due to their relatively large size, the proteinscannot cross the blood-brain barrier and access the most importantneurological site in the body. Alternatively, the direct injection ofneurotrophic factors into the brain or cerebrospinal fluid crudelyovercomes this difficulty but is fraught with technical problems of itsown which have thus far proven intractable. For example, direct infusionof known neurotrophins into the brain has proven impractical as itrequires administration over a period of years to provide therapeuticconcentrations. Further, direct injection into the brain has beenassociated with dangerous swelling and inflammation of the nerve tissueafter a very short period of time. Moreover, such direct injection ofsubstances such as neurotrophic factors into the brain or other nervoustissue is often repugnant to the patient and is thus not acceptable,particularly when it is required repeatedly over a long period of time.

Therefore, there is a need for improved methods of administeringneurotrophic factors or stimulating their production so as to enablenerve growth or regeneration.

Accordingly, it is a general object of the present invention to providemethods and associated compositions for effectively modifying mammalianneurons or neural activity to achieve a variety of beneficial results.

Thus, it is another object of the present invention to provide methodsand associated compositions for treating mammalian neurological diseasesand disorders.

It is yet another object of the present invention to provide methods andassociated compositions for inducing long-term changes in the membranepotential of a mammalian neuron.

It is still yet another object of the present invention to providemethods and associated compositions for inducing the in vivophysiological production of genetically encoded molecules andneurotrophic factors within cells.

It is a further object of the present invention to provide methods andassociated compositions for enhancing the neuritogenic effects ofneurotrophic factors in a physiological environment.

SUMMARY OF THE INVENTION

These and other objects are accomplished by the methods and associatedcompositions of the present invention which, in a broad aspect, providefor the selective inducement of the in vivo genetic expression andresultant production of naturally occurring genetically encodedmolecules including neurotrophic factors, and for the modification ofcellular and neural activity through the treatment of mammalian cellswith at least one carbon monoxide dependent guanylyl cyclase modulatingpurine derivative. As will be appreciated by those skilled in the art,the in vivo activation or derepression of genetic expression and theexemplary modification of neural activity brought about by the methodsof the present invention may be expressed in a variety of forms orcombinations thereof. For example, the treatment of a mammalian cell orneuron through the teachings of the present invention may result indirect administration to the cell of the in vivo expressed moleculethrough the enhanced cellular production of various naturally occurringgenetically encoded neurotrophic factors or in the stimulation of theactivity of those factors and their subsequent effect of naturallyoccurring neuronal development and survival. The methods of the presentinvention may also stimulate the growth, development, and survival ofthe cell or neuron directly without the deleterious effect of prior artneurotrophic factor methodology. Further, the present invention may beused to lower or change the membrane potential of the cell, increasingits plasticity and inducing long term potentiation.

Exemplary carbon monoxide dependent guanylyl cyclase modulating purinederivatives useful for practicing the present invention includeguanosine, inosine pranobex, andN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl)propanamide (also known as4-(3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl)amino) benzoic acidand designated AIT-082) and, unlike prior art compounds, these compoundsmay be administered directly to a patient either orally or throughinjection or other conventional routes.

These exemplary compounds are non-toxic and will cross the blood-brainbarrier as well.

In a further, more specific aspect, the methods and compositions of thepresent invention may be used for the treatment or prophylacticprevention of neurological diseases and disorders, including thosebrought about by disease, age, trauma, or exposure to harmful chemicalagents. By promoting the survival, growth, and development of individualneurons and associated cells, the methods of the present inventionthereby facilitate the regeneration and development of the neuralnetwork and alleviate the manifestations of neural dysfunction. Ofcourse, those skilled in the art will appreciate that pharmaceuticalcompositions may be formulated incorporating effective concentrations ofthe carbon monoxide dependent guanylyl cyclase modulating purinederivatives along with pharmaceutically acceptable excipients andcarriers. These pharmaceutical compositions may be administered orally,topically, or by injection. Moreover, as the active agents used in themethods of the present invention can cross the blood-brain barrier, theydo not have to be injected or infused directly into the brain or centralnervous system.

In yet another aspect, the methods and compositions of the presentinvention may be used to induce long term changes in the membranepotential of the mammalian neuron. These long term potentiation changesmay lead to increased membrane plasticity with a correspondingenhancement of cellular memory. In turn, this enhanced cellular memorymay elevate the mental capacity of the subject leading to fasterlearning and increased retention of material.

Specifically, one aspect of the present invention is a method forselectively and controllably inducing the in vivo genetic expression ofat least one naturally occurring genetically encoded molecule in amammal comprising the step of administering an effective amount of atleast one carbon monoxide dependent guanylyl cyclase modulating purinederivative to the mammal.

In this method, the at least one naturally occurring genetically encodedmolecule can be a molecule that stimulates neuritogenesis. The at leastone naturally occurring genetically encoded molecule that stimulatesneuritogenesis can be selected from the group consisting ofneurotrophins, pleiotrophins, members of the S100 family of EF handcalcium binding proteins, and members of the TGFβ superfamily.Neurotrophins can include nerve growth factor (NGF), NT-3, andbrain-derived neurotrophic factor (BDNF). Pleiotrophins can includebasic fibroblast growth factor (bFGF) and ciliary neurotrophic factor(CNTF). Members of the S100 family of EF hand calcium binding proteinscan include S100β, p11, p9Ka, and calcyclin. Members of the TGFβsuperfamily can include TGFβ₁ and glial line-derived neurotrophic factor(GDNF).

The carbon monoxide dependent guanylyl cyclase modulating purinederivative can be selected from the group consisting of guanosine,inosine pranobex, and a compound of formula (I)

where n is an integer from 1 to 6 or of a salt or prodrug ester of acompound of formula (I) where n is an integer from 1 to 6. Typically,the compound is a compound of formula (I) where n is an integer from 1to 6. Preferably, n is 2 and the compound isN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide.

Typically, the effective amount of the at least one carbon monoxidedependent guanylyl cyclase modulating purine derivative produces atreating concentration of at least 1 μM.

The at least one carbon monoxide dependent guanylyl cyclase modulatingpurine derivative can be orally administered to the mammal.Alternatively, the at least one carbon monoxide dependent guanylylcyclase modulating purine derivative can be administered to the mammalby injection.

The mammal can be a human.

The induction of the in vivo genetic expression of at least onenaturally occurring genetically encoded molecule can occur in astrocytesof the mammal. The induction of the in vivo genetic expression of atleast one naturally occurring genetically encoded molecule can activatethe mitogen-activated protein kinase cascade.

Another aspect of the present invention is a method for theadministration of at least one naturally occurring genetically encodedmolecule to a mammal comprising the step of selectively inducing the invivo genetic expression of the molecule in the mammal through theadministration of an effective amount of at least one carbon monoxidedependent guanylyl cyclase modulating purine derivative to the mammal toraise the concentration of the at least one naturally occurringgenetically encoded molecule in at least one tissue of the mammal andthus cause the administration of the at least one naturally occurringgenetically encoded molecule to the mammal.

Yet another aspect of the present invention is a method for modifyingthe membrane potential of a mammalian neuron comprising the step ofadministering an effective amount of at least one carbon monoxidedependent guanylyl cyclase modulating purine derivative to the mammalianneuron.

The effective amount of the at least one carbon monoxide dependentguanylyl cyclase can be administered to a mammal so that the methodproduces an increased learning capability in the mammal.

Still another aspect of the present invention is a method forselectively and controllably inducing the in vivo genetic expression ofat least one naturally occurring genetically encoded molecule in amammal comprising the step of administering an effective amount of atleast one carbon monoxide dependent guanylyl cyclase modulating guaninederivative to the mammal, the guanine derivative comprising a guaninemoiety linked through its nitrogen-9 atom through a linker to aphysiologically active group. Typically, the linker of the guaninederivative incorporates a hydrocarbyl moiety that includes a carbonylgroup at one end. Preferably, the end of the hydrocarbyl moiety that isterminated with the carbonyl group is linked to the physiologicallyactive group through an amide linkage.

Preferably, the guanine derivative comprises a compound of formula (II)

wherein n is an integer from 1 to 6.

More preferably, in a compound of formula (II), n is 2 and the compoundis N-4-carboxyphenyl-3-(2-amino-6-oxohydropurin-9-yl) propanamide.

Other objects, features and advantages of the present invention will beapparent to those skilled in the art from a consideration of thefollowing detailed description of preferred exemplary embodimentsthereof taken in conjunction with the data expressed in the associatedfigures which will first be described briefly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of murine plasma concentrationfollowing administration of the purine derivative AIT-082 in accordancewith the methods of the present invention;

FIG. 2 is a graphical representation of the effect of atropine, acholinergic antagonist, on memory enhancement in mice by the purinederivative AIT-082;

FIG. 3 is a graphical representation of nerve growth factor mediatedneuritogenic response in neuronal cells grown in vitro with variousconcentrations of the purine derivative AIT-082;

FIGS. 4A, 4B and 4C are graphical comparisons of the effects ofselective inhibitors and the purine derivative AIT-082 on nerve growthfactor mediated neuritogenic response; FIG. 4A shows the neuritogenicresponse of cells grown in the presence of methemoglobin, a carbonmonoxide scavenger; FIG. 4B shows the same response of cells grown inthe presence of methylene blue, a guanylyl cyclase inhibitor FIG. 4Cshows the response of cells grown in the presence of zinc protoporphyrinIX, a carbon monoxide scavenger;

FIGS. 5A and 5B are graphical comparisons of nerve growth factormediated neuritogenic response for cells grown in the presence of thepurine derivative AIT-082 and various concentrations of nitric oxideinhibitors;

FIG. 6 is a graphical comparison of cyclic GMP production in neuronalcells grown in culture with the purine derivative AIT-082 and withoutAIT-082;

FIG. 7 is a graphical representation of the effects of different dosesof the purine derivative AIT-082 on learning as measured in SwissWebster mice using a win-shift memory test;

FIG. 8 is a graphical comparison of the duration of action of the purinederivative AIT-082 measured over time for single doses of 60 mg/kg and30 mg/kg;

FIG. 9 is a graphical comparison of learning abilities of age-inducedmemory deficit Swiss Webster mice treated with the purine derivativeAIT-082 and the drug physostigmine;

FIG. 10 is a graphical comparison of learning abilities of age-inducedmemory deficit C57BL/6 mice treated with the purine derivative AIT-082and the drug physostigmine;

FIG. 11 is a graphical comparison of age-induced memory deficitprophylaxis in mice treated with the purine derivative AIT-082 anduntreated mice;

FIGS. 12A and 12B are graphical comparisons of the production of nervegrowth factor by murine cortical astrocytes in response to the additionof purine derivatives as measured using an ELISA assay; FIG. 12Aillustrates measured nerve growth factor concentrations for neuronsgrown in the presence of different concentrations of guanosinetriphosphate and FIG. 12B illustrates nerve growth factor concentrationsfor cells grown in the presence of various concentrations of guanosine;

FIGS. 13A and 13B are graphical comparisons of the production of variousneurotrophic factor mRNA by murine cortical astrocyte cells grown in thepresence and absence of guanosine at different times; FIG. 13Aillustrates mRNA levels of nerve growth factor (NGF) and FIG. 13Billustrates mRNA levels of fibroblast growth factor (FGF);

FIGS. 14A, 14B and 14C are graphical comparisons of neuritogenicresponses to different concentrations of purine derivative in thepresence and absence of nerve growth factor; FIG. 14A illustratesneuritogenic response to various purine derivatives at differentconcentrations in the presence of nerve growth factor, FIG. 14Billustrates neuritogenic response in the absence of nerve growth factorand FIG. 14C illustrates neuritogenic response to individual purinederivatives and combinations of purine derivatives in the presence andabsence of nerve growth factor;

FIGS. 15A, 15B and 15C are graphical comparisons of nerve growth factormediated neuritogenic responses in neurons grown in the presence ofvarious concentrations of different purine derivatives; FIG. 15Aillustrates neuritogenic response to various concentrations of inosine;FIG. 15B illustrates the same neuritogenic response to variousconcentrations of hypoxanthine and FIG. 15C illustrates the neuritogenicresponse of neuronal cells exposed to different concentrations ofxanthine;

FIG. 16 is a graphical representation of nerve growth factor mediatedneuritogenesis measured for neuronal cells grown at variousconcentrations of the purine derivative AIT-034;

FIG. 17 is a graphical comparison of neuritogenic response of neuronalcells grown at various concentrations of guanosine triphosphate andadenosine triphosphate with and without nerve growth factor;

FIG. 18 is a graphical comparison of nerve growth factor mediatedneuritogenic response to monophosphate, diphosphate, and triphosphatepurine derivatives of guanosine and adenosine;

FIG. 19 is a graphical comparison of cyclic GMP produced in neuronalcells grown in the presence of different concentrations of the purinederivative guanosine;

FIGS. 20A, 20B and 20C are graphical comparisons of nerve growth factormediated neuritogenic responses of cells grown with and without thepurine derivative guanosine in the presence of various concentrations ofthree different inhibitors; FIG. 20A illustrates the neuritogenicresponse of cells grown in the presence of methylene blue, a guanylylcyclase inhibitor, FIG. 20B illustrates the neuritogenic response ofcells grown in the presence of various concentrations of LY83583, alsoan inhibitor of guanylyl cyclase, FIG. 20C illustrates the neuritogenicresponse of cells grown in the presence of various concentrations ofatrial natriuretic factor, a hormone which interacts with guanylylcyclase;

FIG. 21 is a graphical representation of nerve growth factor-mediatedneuritogenic responses for neurons grown in the presence of sodiumnitrate, an inorganic nitric oxide donor;

FIGS. 22A and 22B are graphical comparisons of nerve growth factormediated neuritogenic response of neurons grown in the presence ofnitric oxide donors and scavengers of nitric oxide and carbon monoxide;FIG. 22A shows the neuritogenic response of cells grown in the presenceof various combinations of nitric oxide donors and hemoglobin and FIG.22B shows the neuritogenic response of cells grown in the presence ofvarious combinations of nitric oxide donors and methemoglobin;

FIG. 23 is a graphical comparison showing the nerve growth factormediated neuritogenic response of cells grown in various concentrationsof hemoglobin with or without the purine derivative guanosine;

FIG. 24 is a graphical comparison showing the nerve growth factormediated neuritogenic response of cells grown in various concentrationsof L-nitro arginine methyl ester (L-NAME) with and without the purinederivative guanosine;

FIG. 25 is a graphical comparison of the nerve growth factor mediatedneuritogenic response for cells grown in the presence of variousconcentrations of zinc protoporphyrin IX (ZNPP), an inhibitor of COsynthesis, with and without guanosine;

FIG. 26 is a negative control for the graphical comparison shown in FIG.25 and is a graphical comparison of nerve growth factor mediatedneuritogenic response for cells grown in various concentrations ofcopper protopoiphyrin IX (CUPP), with and without the purine derivativeguanosine;

FIG. 27 is a graphical representation of the nerve growth factormediated neuritogenic response for neuron cells grown in the presence ofvarious concentrations of the purine derivative inosine pranobex.

FIG. 28 is a graph showing the dose-dependent increase of the outflow ofradioactive adenine-based purines for a limited period after exposure toAIT-082;

FIG. 29 is a graph of the effect of AIT-082 on the proportional releaseof radioactively labeled adenine nucleosides and nucleotides from ratcultured astrocytes: (A) ATP, ADP, and AMP; (B) adenosine, inosine, andhypoxanthine;

FIG. 30 is a graph showing the dose response for AIT-082 and guanosineon nerve growth factor and S100β protein release from rat cultureastrocytes: (A) nerve growth factor; (B) S100β protein;

FIG. 31 is a graph showing the time course of AIT-082 induced release ofNGF and S100β protein from rat cultured astrocytes;

FIG. 32 is a graph showing the time course of AIT-082 induced release ofNGF and S100β protein from rat cultured astrocytes;

FIG. 33 is a graph showing the effect of conditioned medium removed fromastrocytes previously treated with AIT-082 on the toxicity induced byNMDA as measured by the number of dead neurons and the production ofLDH;

FIG. 34 is a graph showing the effect of anti-NGF antibody onAIT-induced protection of glial conditioned medium in culturedhippocampal neurons damaged by NMDA;

FIG. 35 is a graph showing the damage to glutamic acid decarboxylase(GAD) activity in rat caudate nuclei caused by administration of NMDA;

FIG. 36 is a photomicrograph showing the serial frontal sections acrossthe extension of the caudate nuclei from a rat locally infused with 200nmoles of NMDA;

FIG. 37 is a graph showing the effect of local administration of AIT-082on NMDA-induced unilateral lesion of rat striatum;

FIG. 38 is a graph showing the effect of systemic administration ofAIT-082 on GAD activity in rat caudate nuclei damaged by NMDA;

FIG. 39 is a photograph of MRI images of rat striatum injected withsaline (A, B), NMDA (C, D), NMDA locally co-injected with AIT-082 (E,F), or NMDA with systemic administration of AIT-082 (G, H);

FIG. 40 is a diagram of the location of the lesions made in the spinalcord of rats to which AIT-082 was administered in Example 37;

FIG. 41 is a graph of the level of niRNA for the neurotrophic factorsbrain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor(CNTF), and NT-3 at the lesion site for animals lesioned orsham-lesioned in Example 37: (a) levels measured 3 days after lesioningor sham-lesioning; (b) levels measured 7 days after lesioning orsham-lesioning;

FIG. 42 is a graph of the levels of mRNA for BDNF, CNTF, and NT-3rostral to the lesion for animals lesioned or sham-lesioned in Example37: (a) levels measured 3 days after lesioning or sham-lesioning; (b)levels measured 7 days after lesioning or sham-lesioning;

FIG. 43 is a graph of the levels of mRNA for BDNF, CNTF, and NT-3 caudalto the lesion for animals lesioned or sham-lesioned in Example 37: (a)levels measured 3 days after lesioning or sham-lesioning; (b) levelsmeasured 7 days after lesioning or sham-lesioning;

FIG. 44 is a graph showing the effect of AIT-082 treatment upon medialseptum cholinergic neurons following fimbria-fornix transections;

FIG. 45 is a graph showing the effect of AIT-082 treatment on BDNFprotein levels in the intact adult rat brain as measured by an ELISAassay as determined in the frontal cortex (F. Cor.), parietal cortex(P.Cor.), hippocampal formation (Hipp.), basal forebrain (B.F.),cerebellum (Cer.), or spinal cord (Cord);

FIG. 46 is a graph showing the effect of AIT-082 treatment on NT-3protein levels in the intact adult rat brain as measured by an ELISAassay as determined in the frontal cortex (F. Cor.), parietal cortex(P.Cor.), hippocampal formation (Hipp.), basal forebrain (B.F.),cerebellum (Cer.), or. spinal cord (Cord);

FIG. 47 is a graph showing the effect of AIT-082 treatment on GDNFprotein levels in the intact adult rat brain as measured by an ELISAassay as determined in the frontal cortex (F. Cor.), parietal cortex(P.Cor.), hippocampal formation (Hipp.), basal forebrain (B.F.),cerebellum (Cer.), or spinal cord (Cord);

FIG. 48 is a graph showing the effect of AIT-082 treatment on NGFprotein levels in the intact adult rat brain as measured by an ELISAassay as determined in the frontal cortex (F. Cor.), parietal cortex(P.Cor.), hippocampal formation (Hipp.), basal forebrain (B.F.),cerebellum (Cer.), or spinal cord (Cord);

FIG. 49 is a graph showing the effect of AIT-082 treatment on BDNFprotein levels in the fimbria-fomix transection-lesioned adult rat brainas measured by an ELISA assay as determined in the frontal cortex (F.Cor.), parietal cortex (P.Cor.), hippocampal formation (Hipp.), basalforebrain (B.F.), cerebellum (Cer.), or spinal cord (Cord);

FIG. 50 is a graph showing the effect of AIT-082 treatment on NT-3protein levels in the fimbria-fomix transection-lesioned adult rat brainas measured by an ELISA assay as determined in the frontal cortex (F.Cor.), parietal cortex (P.Cor.), hippocampal formation (Hipp.), basalforebrain (B.F.), cerebellum (Cer.), or spinal cord (Cord);

FIG. 51 is a graph showing the effect of AIT-082 treatment on GDNFprotein levels in the fimbria-fornix transection-lesioned adult ratbrain as measured by an ELISA assay as determined in the frontal cortex(F. Cor.), parietal cortex (P.Cor.), hippocampal formation (Hipp.),basal forebrain (B.F.), cerebellum (Cer.), or spinal cord (Cord);

FIG. 52 is a graph showing the effect of AIT-082 treatment on NGFprotein levels in the fimbria-fornix transection-lesioned adult ratbrain as measured by an ELISA assay as determined in the frontal cortex(F. Cor.), parietal cortex (P.Cor.), hippocampal formation (Hipp.),basal forebrain (B.F.), cerebellum (Cer.), or spinal cord (Cord);

FIG. 53 is a graph showing the effect of AIT-082 treatment on BDNFprotein levels in the intact aged rat brain as measured by an ELISAassay as determined in the frontal cortex (F. Cor.), parietal cortex(P.Cor.), hippocampal formation (Hipp.), basal forebrain (B.F.),cerebellum (Cer.), or spinal cord (Cord);

FIG. 54 is a graph showing the effect of AIT-082 treatment on NT-3protein levels in the intact adult rat brain as measured by an ELISAassay as determined in the frontal cortex (F. Cor.), parietal cortex(P.Cor.), hippocampal formation (Hipp.), basal forebrain (B.F.),cerebellum (Cer.), or spinal cord (Cord);

FIG. 55 is a graph showing the effect of AIT-082 treatment on GDNFprotein levels in the intact adult rat brain as measured by an ELISAassay as determined in the frontal cortex (F. Cor.), parietal cortex(P.Cor.), hippocampal formation (Hipp.), basal forebrain (B.F.),cerebellum (Cer.), or spinal cord (Cord);

FIG. 56 is a graph showing the effect of AIT-082 treatment on NGFprotein levels in the intact adult rat brain as measured by an ELISAassay as determined in the frontal cortex (F. Cor.), parietal cortex (P.Cor.), hippocampal formation (Hipp.), basal forebrain (B.F.),cerebellum (Cer.), or spinal cord (Cord);

FIG. 57 is a graph showing the dose response of NGF and TGFβ₂ from ratcultured astrocytes as induced by guanosine;

FIG. 58 is a gel electropherogram showing Western blot analysis ofcytosolic proteins from cultured astrocytes to detect production of NGFand TGFβ₂ from rat cultured astrocytes as induced by guanosine: (A) NGF;(B) TGFβ₂;

FIG. 59 is a graph showing the effect of the mitogen-activated proteinkinase cascade and ex novo protein synthesis in the guanosine-inducedeffect: (A) effects of inhibitors of the protein kinase cascadewortmannin and PD098,059; (B) effect of the protein synthesis inhibitorcycloheximide;

FIG. 60 is a gel electropherogram of a Western blot showing theactivation of MAP kinases ERK1 (44 kDa) and ERK2 (42 kDa) by guanosine;

FIG. 61 is a graph showing the dose-response curves of NGF and TGFβ₂release from rat cultured astrocytes as the result of exposure toAIT-082;

FIG. 62 is a gel electropherogram of a Western blot showing thestimulation of synthesis of NGF and TGFβ₂ by AIT-082;

FIG. 63 is a gel electropherogram of a Western blot showing theactivation of MAP kinases ERK1 (44 kDa) and ERK2 (42 kDa) by AIT-082;

FIG. 64 is a graph showing the effect of the MAP kinase cascadeinhibitors wortmannin and PD098,059 or the protein synthesis inhibitorcycloheximide on the effect of AIT-082: wortmannin and PD098,059 (leftpanel); cycloheximide (right panel);

FIG. 65 is a graph showing the experimental scheme for the use ofconditioned medium (CM);

FIG. 66 is a graph showing the effects of NMDA on cortical cells (A) orhippocampal cells (B) as demonstrated by count of dead cells as measuredby Trypan Blue staining (left panel) and also by release of LDH activity(right panel);

FIG. 67A is a graph showing the protection provided by CM alone ortogether with 100 μM AIT-082 for hippocampal cells, together withresults when anti-NGF antibody is added together with CM and AIT-082;and

FIG. 67B is a graph showing the protection provided by CM alone ortogether with 100 μM AIT-082 for hippocampal cells, together withresults when anti-TGFβ₂ antibody is added together with CM and AIT-082.

DETAILED DESCRIPTION

In a broad aspect, the present invention is directed to methods andassociated compositions for use in uniquely treating mammalian cells andneurons to modify cellular or neural activity. More specifically, thepresent invention is directed to the use of effective purine derivativesto modulate the carbon dioxide dependent guanylyl cyclase regulatorysystem within cells or neurons to produce a variety of beneficialresults, including the inducement of in vivo genetic expression ofnaturally occurring neurotrophic factors and the resultant directadministration of such naturally occurring genetically encoded moleculesto a mammal. In exemplary embodiments illustrative of the teachings ofthe present invention, particular purine derivatives were used to inducegenetic expression of encoded molecules, to stimulate neuritogenesis, toenhance neuronal growth and to modify the membrane potential of neuronsto produce increased learning capabilities in mammals. Exemplary studiesand treatments were performed as discussed below using various dosagesand routes of administration of selected exemplary purine derivativesrepresentative of compositions that are effective with the methods ofthe present invention. Of course, those skilled in the art willrecognize that the present invention is not specifically limited to theparticular compositions, dosages or routes of administration detailedbelow.

Depending upon the particular needs of the individual subject involved,the compositions used in the present invention may be administered invarious doses to provide effective treatment concentrations based uponthe teachings of the present invention. What constitutes an effectiveamount of the selected composition will vary based upon such factorsincluding the activity of the selected purine derivative, thephysiological characteristics of the subject, the extent and nature ofthe subject's neurodegradation or disorder and the method ofadministration. Exemplary treatment concentrations which have proveneffective in modifying neural activity range from less than 1 μM toconcentrations of 500 mM or more. Generally, initial doses will bemodified to determine the optimum dosage for treatment of the particularmammalian subject. The compositions may be administered using a numberof different routes including orally, topically, transdermally,intraperitoneal injection or intravenous injection directly into thebloodstream. Of course, effective amounts of the purine derivatives mayalso be administered through injection into the cerebrospinal fluid orinfusion directly into the brain, if desired.

The methods of the present invention may be effected using purinederivatives administered to a mammalian subject either alone or incombination as a pharmaceutical formulation. Further, the purinederivatives may be combined with pharmaceutically acceptable excipientsand carrier materials such as inert solid diluents, aqueous solutions ornon-toxic organic solvents. If desired, these pharmaceuticalformulations may also contain preservatives and stabilizing agents andthe like, as well as minor amounts of auxiliary substances such aswetting or emulsifying agents, as well as pH buffering agents and thelike which enhance the effectiveness of the active ingredient. Thepharmaceutically acceptable carrier can be chosen from those generallyknown in the art, including, but not limited to, human serum albumin,ion exchangers, dextrose, alumina, lecithin, buffer substances such asphosphate, glycine, sorbic acid, potassium sorbate, propylene glycol,polyethylene glycol, and salts or electrolytes such as protaminesulfate, sodium chloride, or potassium chloride. Other carriers can beused.

Liquid compositions can also contain liquid phases either in addition toor to the exclusion of water. Examples of such additional liquid phasesare glycerin, vegetable oils such as cottonseed oil, organic esters suchas ethyl oleate, and water-oil emulsions.

The compositions can be made into aerosol formations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichloromethane, propane, or nitrogen. Other suitable propellants areknown in the art.

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions. These can contain antioxidants, buffers, preservatives,bacteriostatic agents, and solutes that render the formulation isotonicwith the blood of the particular recipient. Alternatively, theseformulations can be aqueous or non-aqueous sterile suspensions that caninclude suspending agents, thickening agents, solubilizers, stabilizers,and preservatives. Compositions suitable for use in methods according tothe present invention can be administered, for example, by intravenousinfusion, orally, topically, intraperitoneally, intravesically, orintrathecally. Formulations of compounds suitable for use in methodsaccording to the present invention can be presented in unit-dose ormulti-dose sealed containers, in physical forms such as ampules orvials.

The methods of the present invention provide for the long termmodification of various types of cellular or neural activity includingthe in vivo production of naturally occurring genetically encodedmolecules such as neurotrophic growth factors (including neurotrophins,pleiotrophins and cytokines), directly administering such in vivoproduced molecules, enhancing the effects of these neurotrophic factors,and the stimulation of cell growth and development. Further, the methodsof the present invention may be used to promote neuritogenesis, to formcollateral nerve circuits, to enhance the production of cyclic purinenucleotides, to enhance synapse formation and to alter the membranepotential of the neuron. These effects may be extremely beneficial intreating neurodegeneration and increasing learning capacity.

For obvious practical and moral reasons, initial work in humans todetermine the efficacy of experimental compositions and methods withregard to such afflictions is unfeasible. Accordingly, in the earlydevelopment of any drug or therapy it is standard procedure to employappropriate animal models for reasons of safety and expense. The successof implementing laboratory animal models is predicated on theunderstanding that the cellular or neurophysiology of mammals issimilar. Thus, a cellular or neurotropic response in a member of onespecies, for example, a rodent, frequently corresponds to the samereaction in a member of a different species, such as a human. Only afterthe appropriate animal models are sufficiently developed will clinicaltrials in humans be carried out to further demonstrate the safety andefficacy of a therapeutic agent in man.

With regard to neurodegenerative diseases and disorders and to theirclinical effects, the mouse model closely resembles the human pathologyof these conditions in many respects. Accordingly, it is well understoodby those skilled in the art that it is appropriate to extrapolate themouse or “murine” model to humans and to other mammals. As with humans,mice are susceptible to learning disorders resulting from neuronaldegradation, whether due to traumatic injury, age, disease or harmfulchemical agents. Just as significantly, neurotropic factors appear toact in substantially the same manner in a murine model as they do inhumans with remarkably similar neuronal reactions. Accordingly, forpurposes of explanation only and not for purposes of limitation, thepresent invention will be primarily demonstrated in the exemplarycontext of mice as the mammalian subject. Those skilled in the art willappreciate that the present invention may be practiced with othermammalian subjects, including humans, as well.

As will be shown by the data herein, several purine derivatives havebeen found to work effectively in accordance with the teachings of thepresent invention. In particular, the data shows that guanosine appearsto work well in stimulating the production of neurotrophic factors andenhancing neuritogenesis. Similarly another exemplary purine derivative,N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, also known as4-(3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl)amino) benzoic acid(AIT-082) has been shown to stimulate the in vivo activation orderepression of naturally occurring genes and the resultant productionof naturally occurring genetically encoded molecules such asneurotrophic factors; and to increase neuritogenesis, enhance theeffects of neurotrophic factors and alter the membrane potential ofneurons thereby facilitating long term potentiation of the cells.AIT-082 is disclosed in U.S. Pat. No. 5,091,432 issued Feb. 25, 1992 toa co-inventor of the present application and incorporated herein byreference. Yet another exemplary composition which has been shown to besuitable for use in the present invention is inosine pranobex orisoprinosine. Inosine pranobex, a mixture of inosine anddimethylaminoisopropanol acetamidobenzoate (DIP-PacBa) at a 1:3 molarratio was found to enhance neuritogenesis and the effects ofneurotrophic factors in vitro. The different embodiments of the presentinvention presented above demonstrate the applicability of using variouspurine derivatives to modify neural activity through modulating thecarbon monoxide dependent guanylyl cyclase system.

Exemplary preferred embodiments of the methods of the present inventioninvolve the treatment of cells or neurons with AIT-082 or4-(3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl)amino) benzoic acid.AIT-082 is a unique derivative of the purine hypoxanthine containing apara-aminobenzoic acid moiety. It is rapidly absorbed after oraladministration and, after crossing the blood brain barrier, enters thebrain unchanged. It may be detected at levels as high as 3.3 ng/mg braintissue 30 minutes after oral administration. AIT-082 induces the in vivogenetic expression of naturally occurring genetically encoded moleculesincluding neurotrophic factors. As a result, it directly administersthese compounds to the treated cells and stimulates neurite outgrowthfrom neuronal cells when added alone to the cultures as well asenhancing the neuritogenic effects of neurotrophic factors such as nervegrowth factor (NGF). More importantly, AIT-082 enhances working memoryin old, memory deficient mice after intraperitoneal and oraladministration. The neuritogenic activity of AIT-082 is inhibited byhemoglobin, by Methylene Blue, and by ZnPP, all scavengers of CO, butnot by CuPP or by other inhibitors of nitric oxide synthase. Screeningtests for in vitro activity at known neurotransmitter and neuromodulatorreceptors were negative.

A further understanding of the present invention will be provided tothose skilled in the art from the following non-limiting examples whichillustrate exemplary protocols for the identification, characterizationand use of purine derivatives in accordance with the teachings of thepresent invention.

In particular, one aspect of the present invention is a method forselectively and controllably inducing the in vivo genetic expression ofat least one naturally occurring genetically encoded molecule in amammal comprising the step of administering an effective amount of atleast one carbon monoxide dependent guanylyl cyclase modulating purinederivative to the mammal.

In this method, the at least one naturally occurring genetically encodedmolecule can be a molecule that stimulates neuritogenesis. The at leastone naturally occurring genetically encoded molecule that stimulatesneuritogenesis can be selected from the group consisting ofneurotrophins, pleiotrophins, members of the S100 family of EF handcalcium binding proteins, and members of the TGFβ superfamily.

The induction of the in vivo genetic expression of at least onenaturally occurring genetically encoded molecule can occur in astrocytesof the mammal, as demonstrated below in the Examples. This inductioncauses astrocytes to produce factors that exert a neuroprotective effectagainst agents such as excitotoxins.

The induction of the in vivo genetic expression of at least onenaturally occurring genetically encoded molecule can activate themitogen-activated protein kinase cascade, as shown below in theExamples. The protein kinases activated in this cascade include ERK1 (44kilodaltons) and ERK2 (42 kilodaltons).

Neurotrophins can include nerve growth factor (NGF), NT-3, andbrain-derived neurotrophic factor (BDNF).

Functional nerve growth factor is a non-covalently linked parallelhomodimer. The structure of nerve growth factor consists of threeanti-parallel pairs of β-strands together forming a flat surface throughwhich the two subunits associate.

The amino acid sequence for human nerve growth factor and mouse nervegrowth factor is known. This molecule is described in R. E. Callard & A.J. H. Gearing, “The Cytokine Facts Book” (Academic Press, London, 1994),pp. 191-198, incorporated herein by this reference.

The growth factor NT-3 also promotes the survival and outgrowth ofneural crest-derived sensory and sympathetic neurons. The structure ofthis molecule is known; its amino acid sequence is identical in thehuman and mouse. The structure has 60% β-sheet secondary structure andexists as a tightly linked homodimer. NT-3 is described in R. E. Callard& A. J. H. Gearing, “The Cytokine Facts Book” (Academic Press, London,1994), pp. 199-200, incorporated herein by this reference.

Brain-derived neurotrophic factor also promotes the survival of neuronalpopulations located either in the central nervous system or directlyconnected to it. It helps to maintain neurons and their differentiatedphenotype in the adult. The amino acid sequence is known for human andmouse BDNF. The molecule has 70% β-sheet secondary structure and isexpressed as a tightly associated homodimer. Properties of this moleculeare described in R. E. Callard & A. J. H. Gearing, “The Cytokine FactsBook” (Academic Press, London, 1994), pp. 99-100, incorporated herein bythis reference.

Pleiotrophins can include basic fibroblast growth factor (bFGF) andciliary neurotrophic factor (CNTF).

Basic fibroblast growth factor (bFGF), also known as FGF-2, is a proteinof 155 amino acids for the human factor. The molecule has an isoelectricpoint of about 9.6. The molecule is composed entirely of a β-sheetstructure with a threefold repeat of a four-stranded antiparallelβ-meander which forms a barrel-like structure with three loops. Theamino acid sequences for human and mouse bFGF are known. There is nosignal sequence, but a truncated form can arise by cleavage betweenresidues 9-10. Further information about bFGF is given at R. E. Callard& A. J. H. Gearing, “The Cytokine Facts Book” (Academic Press, London,1994), pp. 120-123, incorporated herein by this reference.

Ciliary neurotrophic factor also promotes the survival anddifferentiation of neuronal cells. CNTF has no homology with NGF, DDNF,and NT-3. The absence of a signal peptide and N-linked glycosylationsites in CNTF is consistent with it being a cytosolic protein. Thethree-dimensional structure of CNTF is not known, but it has significanthomologies with other cytokines, such as IL-6, LIF, oncostatin M, andG-CF. It is thought that these molecules share a four-helix bundlestructure. The amino acid sequences of human CNTF and rat CNTF areknown. Although these sequences are similar, they are not identical.Further information about CNTF is given at R. E. Callard & A. J. H.Gearing, “The Cytokine Facts Book” (Academic Press, London, 1994), pp.104-105, incorporated herein by this reference.

Members of the S100 family of EF hand calcium binding proteins caninclude S100β, p11, p9Ka, and calcyclin. All of these proteins includetwo EF-hand domains and bind calcium ions.

Members of the TGFβ superfamily can include TGFβ₁ and glial line-derivedneurotrophic factor (GDNF).

TGFβ₁ is a dimeric protein of two identical subunits, each with 112amino acids, linked by disulfide bonds. The amino acid sequence of humanTGFβ₁ is known. There is greater than 98% homology between thefunctional regions of the human and mouse proteins. Further informationabout TGFβ₁ is given in R. E. Callard & A. J. H. Gearing, “The CytokineFacts Book” (Academic Press, London, 1994), pp. 235-236, incorporatedherein by this reference.

GDNF is a glycosylated disulfide-bonded homodimer that promotes survivaland morphological differentiation of dopaminergic neurons. Human and ratGDNF have 93% homology. The mature molecule has 134 amino acids and iscleaved from a precursor of 211 amino acids.

The carbon monoxide dependent guanylyl cyclase modulating purinederivative can be selected from the group consisting of guanosine,inosine pranobex, and a compound of formula (I)

where n is an integer from 1 to 6 or of a salt or prodrug ester offormula (I) where n is an integer from 1 to 6. Typically, the compoundis a compound of formula (I) where n is an integer from 1 to 6.Preferably, n is 2 and the compound isN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, as describedabove.

Salts and prodrug esters are well known in the art. Prodrug estersrelease an active compound by hydrolysis of the ester linkage of theprodrug ester.

In addition to the compounds recited above, a number of other guaninederivatives are useful in the methods of the present invention. Theseare bifunctional compounds that comprise a guanine moiety linked throughits nitrogen-9 atom through a linker to a physiologically active group.The length of the linker is chosen so that both the guanine moiety andthe physiologically active group can bind to two different receptors.Although a large number of linkers can be used to covalently link theguanine moiety and the physiologically active group, a particularlypreferred linker incorporates a hydrocarbyl moiety that includes acarbonyl group at one end. The carbonyl group can be present as part ofa substituted aldehyde residue, as part of an ester moiety, or part ofan amide moiety. Preferably, the hydrocarbyl moiety is saturated andunbranched. The end of the hydrocarbyl moiety that is terminated withthe carbonyl group is linked to the physiologically active group, suchas through an amide linkage. A preferred length of the hydrocarbylmoiety is two carbon atoms; this length does not include thefunctionalized carbon atom that contains the carbonyl group. The lengthof the linker can be varied according to the physiologically activegroup covalently linked to the guanine moiety.

The guanine derivatives that are useful in methods according to thepresent invention include compounds of formula (II), as well as otherguanine derivatives.

A 9-substituted guanine derivative particularly useful in methodsaccording to the present invention is a compound in which thephysiologically active group is a p-aminobenzoic acid analogue. A9-substituted guanine derivative according to the present inventionincorporating a p-aminobenzoic acid analogue has formula (II) wherein nis an integer from 1 to 6.

Preferably, n is 2 and the compound isN-4-carboxyphenyl-3-(2-amino-6-oxohydropurin-9-yl) propanamide.

Analogues of compounds of formula (II) in which the phenyl group of thep-aminobenzoic acid analogue is substituted are also useful in methodsaccording to the present invention.

Typically, the effective amount of the at least one carbon monoxidedependent guanylyl cyclase modulating purine derivative produces atreating concentration of at least 1 μM.

The at least one carbon monoxide dependent guanylyl cyclase modulatingpurine derivative can be orally administered to the mammal.Alternatively, the at least one carbon monoxide dependent guanylylcyclase modulating purine derivative can be administered to the mammalby injection.

The mammal can be a human.

Another aspect of the present invention is a method for theadministration of at least one naturally occurring genetically encodedmolecule to a mammal comprising the step of selectively inducing the invivo genetic expression of the molecule in the mammal through theadministration of an effective amount of at least one carbon monoxidedependent guanylyl cyclase modulating purine derivative to the mammal toraise the concentration of the at least one naturally occurringgenetically encoded molecule in at least one tissue of the mammal andthus cause the administration of the at least one naturally occurringgenetically encoded molecule to the mammal.

Methods for synthesis of suitable compounds for use in methods accordingto the present invention are described, for example, in U.S. Pat. No.5,091,432 to Glasky, incorporated herein by this reference. In general,such methods comprise the steps of: (1) synthesis of an appropriatelysubstituted purine moiety with a 6-amino group linked to an aliphaticlinker in which the linker is terminated with a carboxyl group protectedas with an alkyl ester; (2) converting the 6-amino group to a 6-oxogroup by oxidation, such as with sodium nitrite; (3) hydrolyzing thealkyl ester (or other analogous protecting group) to yield a carboxylicacid; (4) activating the free carboxylic acid by converting it to anitrophenyl ester; (5) reacting the nitrophenyl ester with ap-aminobenzoate moiety protected with an ethyl ester; and (6)hydrolyzing the ethyl ester protecting the p-aminobenzoate moiety toproduct the final product. Other reaction sequences are also known inthe art.

EXAMPLE 1 Plasma Levels of AIT-082in Mice

Adult C57BL/6 mice were administered 30 mg/kg of AIT-082 in saline i.p.The animals were sacrificed by decapitation at 30, 45, 60 and 90 minutesafter administration of AIT-082. Blood was collected in heparinizedtubes, mixed and centrifuged at 2000 rpm for 15 minutes. The plasmasupernatant was removed and stored at −70° C. until analysis. A highpressure liquid chromatography system was developed for the analyticalmeasurement of AIT-082 in plasma and brain tissue. The assay developedwas selective for AIT-082 in the presence of a number of closely relatedpurine molecules. The sensitivity of the method was 0.1 microgram ofAIT-082 per ml of plasma and 0.1 microgram of AIT-082 per milligram ofbrain tissue (wet weight).

The results of these determinations graphically represented in FIG. 1where plasma levels of AIT-082 are provided at 5, 10, 15, 20, 30, 60,90, and 120 minutes after administration of 30 mg/kg i.p. to C57BL/6mice. From the data, it was estimated that the blood level of AIT-082reached its peak at approximately 15 minutes.

EXAMPLE 2 AIT-082 Crosses the Blood Brain Barrier

Brain tissue was analyzed from two animals receiving 30 mg/kg i.p. ofAIT-082 and sacrificed 30 minutes after drug administration. The brainswere rapidly removed and chilled on ice. Brain tissue was dissected intocortex and remainder of the brain. Brain tissue (approx. 250-300 mg wetweight) was homogenized with 5.0 ml of saline using a Brinkman Polytrontissue grinder and stored at −70° C. until analysis. Brain homogenateswere deproteinized by ultrafiltration through Gelman Acrodisc filters;first through a 1.2 micron filter and then through a 0.2 micron filter.A 30 ml sample was injected into the HPLC for analysis as above. Astandard curve was prepared by the addition of known quantities ofAIT-082 to brain homogenates from untreated animals. Analysis of thebrain tissue indicated that AIT-082 was detected in both the cortexsample and the remaining brain samples from both animals. The resultsare shown directly below in Table A.

TABLE A Brain Tissue Levels of AIT-082 Brain Sample Brain weight Levelof AIT-082 # Region (mg) (ng/mg brain tissue) S3 Cortex 181 2.8 S3Remainder 153 3.3 S4 Cortex 146 3.4 S4 Remainder 217 2.3

This demonstration of the presence of AIT-082 in the brain tissue after30 minutes is critical in that it indicates that AIT-082 crosses theblood-brain barrier without degradation.

EXAMPLE 3 AIT-082 Interacts with the Cholinergic System

Because of the finding that there is a severe loss of cholinergicneurons in the hippocampus in Alzheimer's disease patients, there hasbeen considerable interest in the effect on memory of compounds whichalter the activity of this system. Support for the cholinergichypothesis of memory comes from studies using lesions or a stroke model.Lesions of the CA1 region of the hippocampus appear to specificallydisrupt working memory. In the stroke model, occlusion of the vertebraland carotid arteries (30 minutes) produces specific cell loss in the CA1region of the hippocampus and a loss of working memory. In these modelsin aged rats, physostigrnine, a cholinesterase inhibitor, has been shownto improve memory. THA, another drug which increases cholinergicfunction, was shown to improve memory in aged monkeys. The observationthat AIT-082 improves memory in the same general manner as physostigmineand THA raises the question of whether AIT-082 might have some effect onthe cholinergic system.

To elucidate the mechanisms by which AIT-082 improves memory, attemptswere made to block its actions by co-administration of the short-actingcholinergic antagonist atropine to mice and subjecting them to simplelearning tests. Atropine reportedly has the ability to block the effectsof physostigmine and THA. Mice were injected with AIT-082 (30 mg/kg) 2hr prior to testing on days 1 through 4. Atropine (0.5 mg/kg) wasinjected ½ hour prior to testing or 1.5 hours after AIT-082 on day 3only. All injections were i.p. After a reference run to determine wherethe reward was placed in a T-maze, the mice were retested to determineif they could remember the location of the reward. The percentage ofcorrect responses is graphically represented in FIG. 2.

FIG. 2 demonstrates that atropine blocked the memory enhancing activityof AIT-082 on day 3 and that the effect was transient since the memoryenhancing effects of AIT-082 reappeared on day 4 when no atropine wasadministered. This observation suggests that a cholinergic mechanism maybe involved in the action of AIT-082.

EXAMPLE 4 Effect of AIT-082 on Acetylcholine Receptors

The interaction of AIT-082 with acetylcholine receptors was determinedby interference with the binding of QNB (3-quinuclidinyl benzilate) inmouse tissue using the method of Fields (J. Biol. Chem. 253(9):3251-3258, 1978). There was no effect of AIT-082 in this assay.

In the study, mice were treated with AIT-082 at 30 mg/kg 2 hours priorto sacrifice, decapitated and the tissue processed to obtain membranescontaining the acetylcholine receptors. When these tissues were assayedin vitro, there was no effect of AIT-082 on affinity (Kd) for QNB whenAIT-082 was administered under the same conditions as utilized intesting for effects on memory. There was a change in the number ofreceptors (B max) in cortex and striatum, with the cortex showing adecrease and the striatum an increase in acetylcholine binding sites.These data are consistent with the hypothesis that there is an increasedinput to the cortex as a result of AIT-082 being administered to theanimals. Typically, an increased input will result in down regulation ofthe receptors.

EXAMPLE 5 Effect of AIT-082 on Receptor Ligand Binding in vitro

AIT-082 was evaluated for its ability to inhibit ligand binding to 38isolated receptors. The receptors screened and their ligands were:

Adenosine

Amino Acids:

Excitatory Amino Acids (glycine, kainate, MK-801, NMDA, PCP, quisqualateand sigma);

Inhibitory Amino Acids (benzodiazepine, GABA-A, GABA-B, and glycine)

Biogenic Amines (doparnine-1, dopamine-2, serotonin-1, serotonin-2)

Calcium Channel Proteins (nifedipine, omegaconotoxin, chloride,potassium)

Peptide Factors (ANF, EGF, NGF)

Peptides: (angiotensin, arg-vasopressin-V1 and V2, bombesin, CCK centraland peripheral, neurotensin, NPY, somatostatin, substance k, substanceP, VIP)

Second Messenger Systems:

Adenyl Cyclase

Protein Kinase (phorbol ester and inositol triphosphate)

The testing was conducted under contract at Nova Labs (Baltimore, Md.).AIT-082 had no activity in any of the in vitro assays conducted.

Accordingly, while AIT-082 acts through the cholinergic nervous system(atropine blocks its activity), AIT-082 appears to act through amechanism that does not involve direct interaction with acetylcholinereceptors. It is of importance to note that in vitro, AIT-082 does notbind to the adenosine receptor.

AIT-082 was evaluated in a series of psychopharmacological tests thatwere established in order to more fully evaluate the scope of itscentral nervous system activity. Among the tests utilized were:

(a) motor coordination, by the accelerating Roto-Rod treadmill,

(b) exploratory and home cage locomotor activity, by the Stoeltingactivity monitor,

(c) anxiolytic activity, by the elevated Plus maze, and

(d) nocioception.

AIT-082 was compared with standard reference drugs.

EXAMPLE 6 AIT-082 Increases Motor Coordination in Mice

Motor coordination was measured using an accelerating Roto-Rod treadmillfor mice (Ugo Basile Co.). At various times after treatment with salineor drug, mice were placed on the Roto-Rod, which accelerates to maximumspeed over a 5 minute period. The time in seconds at which the subjectfalls off was recorded in Table B directly below. Each animal was tested3 times and the mean time was recorded.

TABLE B Effect of AIT-082 on Roto-Rod Performance AIT dose (mg/kg) Time(sec) Control  123 ± 64 0.005  162 ± 93 0.05 207* ± 73 0.5 184* ± 7630.0 187* ± 68 60.0 229* ± 80 *p < 0.05, t-test vs controls

Subjects receiving AIT-082 showed improved motor coordination byremaining on the roto-rod for longer periods of time when compared tocontrol (saline) or low doses (0.005 mg/kg).

EXAMPLE 7 AIT-082 Does not Inhibit Exploratory Activity

To measure exploratory behavior, subjects received saline or AIT-082administration, were placed in a novel large cage (25×48×16 cm, W×L×H),and movement was measured at one-minute intervals for 30 minutes. Thelarge cage (San Diego Instruments, San Diego, Calif.) was equipped withvertical detectors and rearing movements were also recorded. No effectswere noted with respect to exploratory activity indicating that thesubjects were not incapacitated.

EXAMPLE 8 AIT-082 Does not Inhibit Locomotor Activity

To measure home cage locomotor activity, the home cage was placed on aplatform of an activity monitor (Stoelting Instruments). Home cagelocomotor activity movements were recorded at one minute intervals for15 minutes. Subjects received saline or AIT-082 and were returned totheir home cages. Ten minutes after injection, the home cage wasreplaced on the platform of the activity monitor. Home cage locomotoractivity movements were recorded at one minute intervals for 30 minutes.During the first five minutes, grooming activity was also monitored andrecorded. The results are shown in Table C directly below.

TABLE C Effect of AIT-082 on Locomotor Activity Movements AIT dose (mean± S.D.) (mg/kg) Pre-drug Post-drug Difference Control 1633 ± 434 1385 ±492 248 ± 492 0.005 1884 ± 230 1375 ± 563 509 ± 429 0.05 1718 ± 606 1508± 456 209 ± 340 0.5 1610 ± 349 1320 ± 689 290 ± 435 30.0 1440 ± 264 1098± 189 342 ± 267 60.0 1690 ± 223 634* ± 223 1056* ± 154  *p < 0.05, t =test vs controls

As shown by the data in Table C, at the high dose (60 mg/kg), subjectsmay have become more habituated to their environment and exhibited lessmovement after treatment with AIT-082. Otherwise, no effects were noted.

EXAMPLE 9 AIT-082 Does not Substantially Increase Anxiety

A Plus maze was constructed of black plexiglass consisting of twoopposite-facing open arms (30×5 cm, L×W) and two opposite facing closedarms (30×5×15 cm, L×W×H). The walls of the closed arms were clearplexiglass and the four arms were connected by a central area 5×5 cm.The entire Plus maze was mounted on a base 38 cm above the floor.Testing consisted of placing the subject at one end of one of the openarms. The time the subject took to leave the start position (the first10 cm of the open arm) was recorded. The time it took for the subject toenter halfway into one of the closed arms was also recorded. When thesubject arrived at the half-way point in the closed arm, thethree-minute test session began. During the three-minute test session,the number of times the subject entered the open arms was recorded. Anentry was defined as placing at least two paws onto the platform of theopen arm. There was a slight anxiogenic effect of AIT-082 at 30 mg/kg,but this was not observed at a higher dose (60 mg/kg) or at the lowerdoses (0.005 to 0.5 mg/kg).

EXAMPLE 10 AIT-082 Does not Effect Nocioception

Mice were placed on an electric hot plate (Ominitech) at 55° C. and thelatency time until the subject licked his hind paw was measured. Ifthere was no response by 45 seconds, the trial was terminated. By thistest there was no effect of AIT-082 on nocioception.

EXAMPLE 11 AIT-082 is not Toxic

Preliminary acute toxicity tests in rats and mice of AIT-082 havedemonstrated that the LD50 is in excess of 3000 mg/kg when administeredby the oral or intraperitoneal route. AIT-082 has been evaluated underPanlabs's General Pharmacology Screening Program (Panlabs, 11804 NorthCreek Parkway South, Bothell, Wash. 98011) and the results indicated anabsence of any toxicity when measured in their standard profile of 79different test systems.

By the nature of the chemical structure of AIT-082, it is notanticipated that the compound will be metabolized into any toxicmetabolites.

In conclusion, there were few deleterious effects of AIT-082 on avariety of psychopharmacological tests except for a slight anxiogeniceffect at one dose. There was an increase in motor coordination(roto-rod test) over a range of doses (0.05 to 60 mg/kg) and possibly alearning or habituation effect at one dosage (60 mg/kg) in the locomotortest.

Following psychopharmacological characterization of this exemplarycompound, further studies were conducted to demonstrate the neurogeniceffects of the present invention.

EXAMPLE 12 AIT-082 Promotes Neuritogenesis in PC12 Cells

Much of the work performed in the characterization of the compounds ofthe present invention involved the use of PC12 cells. These cells arederived from a rat pheochromocytoma and when grown in the presence ofNGF, extend neurites, cease cell division and assume manycharacteristics of sympathetic neurons. When cultured in the absence ofnerve growth factor (NGF), few PC12 cells have neurites greater than onecell diameter. Addition of saturating concentrations of NGF for 48 hoursstimulates neurite outgrowth in about 20-35% of the cells. Because theyconstitute a homogeneous population of neuronal-like cells, withoutcontaminating astroglia type cells, it is possible to study the directeffects of the purine based compounds on neurite outgrowth in thesecells.

To demonstrate neuronal modification by the exemplary compounds of thepresent invention, a dose response curve of AIT-082 was generatedmeasuring the stimulation of neuritogenesis in PC12 cells. Cellscultured in RPMI 1640 with 1.5% horse serum and 1.5% fetal bovine serumwere re-plated onto poly-ornithine coated 24-well culture plates(2.5×10⁴ cells per well). AIT-082 and NGF were added to the variouscultures immediately upon plating. After 48 hours, medium was removedand the cells irnmediately fixed in 10% formalin and PBS for 10 minutes.Cells and neurites were counted within 2 days of fixation.

A neurite was defined as a process extending from the cell at least 1cell body diameter in length and displaying a growth cone at its tip.For each treatment, 2 representative microscope fields were counted fromeach of 6 sister cultures receiving identical treatments. The totalnumber of cells counted per well (approximately 100 cells) and the totalnumber of cells containing neurites in each well were used to determinefraction of neurite-bearing cells. The mean values (±SEM) were thendetermined for each of the treatments. To facilitate comparison neuriteoutgrowth was expressed relative to the proportion of cells bearingneurites in the presence of NGF alone (NGF=100%). The effects ofcompounds with and without NGF were compared by analysis of variance(ANOVA) followed by Tukey's test for significance.

The results are shown in FIG. 3 where the curve represents differentlevels of AIT-082 plus saturating concentrations (40 ng/ml) NGF. Thecenter horizontal line represents control values for cells cultured inthe presence of 40 ng/ml NGF alone. Upper and lower horizontal lines areindicative of confidence limits of NGF alone as determined usingstandard statistical methods.

As shown in FIG. 3, AIT-082 stimulates neuritogenesis and enhancesNGF-stimulated neuritogenesis in PC12 cells at low concentrations (1mM). Analysis of the data shows that AIT-082 was as effective as NGF inpromoting neuritogenesis in PC12 cells and enhanced the optimal effectsof NGF by 30%. For the purposes of comparison, and as will be discussedin more detail below, inosine and hypoxanthine are weakly effective instimulating neuritogenesis and in enhancing NGF-stimulatedneuritogenesis in PC12 cells but are effective at lower concentrationsof 30-300 nM. Guanosine produces a significant effect similar to AIT-082but at a higher concentration of 30-300 mM.

EXAMPLE 13 Effect of Inhibitors of AIT-082 Neuritogenesis

Age-related memory loss has been associated with loss of NGF-dependentbasal forebrain neurons. It can be ameliorated by i.c.v. infusion ofNGF. The effect of AIT-082 on neuritogenesis alone and with NGF werestudied using the protocol of Example 12. In order to study themechanism by which AIT-082 exerts its effects, a series of experimentswas conducted in which inhibitors were utilized to block or modifyspecific biochemical processes. All of the cultures contained NGF atoptimal dose (40 ng/ml) so the series without AIT-082 added representedthe effect of the inhibitors on NGF activity. Where indicated, AIT-082was added at 10 mM, its apparent, presently understood, optimal dose.Three selective inhibitors were used.

The results of these studies are shown below in Table D below, and FIGS.4A, 4B, and 4C graphically present the proportion of cells bearingneurites after 48 hours of culture under the conditions indicated. Thebaseline value was cells grown without NGF or AIT-082.

TABLE D Effect of AIT-082 and Selective Inhibitors on NeuritogenesisAlone and with NGF Concen- AIT-082 AIT-082 + Inhibitor tration alone¹NGF alone NGF None 0.2 ± 0.02  0.2 ± 0.02 0.26 ± 0.01 Methemoglobin 0 0.2 ± 0.02 0.26 ± 0.01 1 μM  0.2 ± 0.02 0.17 ± 0.02 Methylene Blue 0 0.2 ± 0.02 0.26 ± 0.01 5 μM 0.24 ± 0.03 0.10 ± 0.01 Zn ProtoporphyrinIX 0 0.20 ± 0.02 0.26 ± 0.01 1 μM 0.22 ± 0.03 0.13 ± 0.01 ¹Proportion ofcells bearing neurites

Methemoglobin (MHb) captures and removes nitric oxide (NO) and carbonmonoxide (CO) from the culture media. Mh had no effect on NGF activitybut inhibited the action of AIT-082, implying that either NO or CO isinvolved in the action of AIT-82.

Methylene blue (MB) inhibits soluble guanylyl cyclase, the enzyme whichproduces cyclic GMP (cGMP), an intracellular substance which, aspreviously discussed, is involved in the second messenger system ofnerve impulse transmission. MB had no effect on NGF activity butinhibited the action of AIT-082, implying that guanylyl cyclase isinvolved in the mechanism of action of AIT-082.

Zinc protoporphyrin IX (ZPP) is an inhibitor of heme oxygenase 2, whichproduces carbon monoxide. ZPP had no effect on NGF activity butinhibited the action of AIT-082, implying that the production of carbonmonoxide is involved in the mechanism of action of AIT-082.

EXAMPLE 14 Effect of Nitric Oxide Inhibitors on AIT-082

Nitric oxide is produced by the action of the enzyme nitric oxidesynthetase (NOS). Two chemicals that have been shown to selectivelyinhibit NOS are N-nitro-L-arginine methyl ester (L-NAME) andN-nitro-L-arginine (NOLA). Different levels of these chemicals wereadministered simultaneously with AIT-082 and neuritogenesis in PC12 wasmeasured using the protocol of Example 12. The results for L-NAME arepresented in Table E while the results for NOLA are presented in TableF. Both tables are shown directly below with graphical representationsof the data presented in FIGS. 5A and 5B.

TABLE E The Effect of L-NAME on Neuritogenesis Concentration of L-NAME(μM) AIT-082 None 0.1 1.0 10.0  0 0.246 ± 0.017 0.259 ± 0.027 0.257 ±0.013 0.251 ± 0.013  10 μM 0.254 ± 0.008 0.220 ± 0.010 0.302 ± 0.0270.254 ± 0.018 100 μM 0.309 ± 0.027 0.257 ± 0.016 0.232 ± 0.019 0.289 ±0.006

TABLE F The Effect of NOLA on Neuritogenesis Concentration of NOLA (μM)AIT-082 None 0.1 1.0 10.0  0 0.246 ± 0.017 0.259 ± 0.009 0.311 ± 0.0160.305 ± 0.017  10 μM 0.254 ± 0.008 0.277 ± 0.016 0.312 ± 0.029 0.298 ±0.019 100 μM 0.309 ± 0.027 0.279 ± 0.027 0.295 ± 0.028 0.310 ± 0.023

As shown by the data in Tables E and F, neither of these inhibitors ofNOS was effective in blocking the effect of AIT-082 on neuritogenesis.These results indicate that NO was not involved in the mechanism ofaction of AIT-082.

EXAMPLE 15 Effect of AIT-082 on cGMP Levels in PC-12 Cells

To demonstrate CO-dependent guanylyl cyclase modification, cyclicguanosine monophosphate (cGMP) levels in PC12 cells were measuredfollowing addition of AIT-82. Initially, PC-12 cells were primed with 40ng/ml NGF for 3 days in low serum medium (1.5% horse serum+5% fetal calfserum). Cells were seeded onto assay plates in low serum mediumcontaining 40 ng/ml NGF and incubated for 1 hour. The medium was changedto low arginine medium (80 μM) with no serum and NGF and papaverine (100mM) where indicated. Test compounds were added for the indicated timeand the reaction was stopped by adding 5% TCA containing 10,000 dpm of³H-cGMP. cGMP was assayed by the radioimmunoassay method of Maurice(Mol. Pharmacol. 37: 671-681, 1990). TCA was purified by adding powderedcharcoal (5 g) and filtering the mixture through Whatman #1 paper. Thisremoved contaminants in the TCA that otherwise interfere with theradioimmunoassay (RIA) of cGMP.

It was necessary to purify the cGMP from cAMP and other contaminantsbefore radioimmunoassay since these other materials can interfere withthe assay. Briefly, the TCA solution was applied to Dowex columns(50W-8X, 200-400 mesh) and eluted. A neutral alumina column was thenplaced under each Dowex column. The cGMP was eluted from the Dowexcolumns into neutral alumina columns by adding 4 mL of 0.05 M HCl toeach Dowex column. The neutral alumina columns were then sequentiallyrinsed with 2 ml of HCl, 4 mL water and finally with 0.2 M sodiumacetate (pH 6.2). The cGMP collected for the RIA eluted in 1 ML ofsodium acetate with a recovery between 50-65%. The cGMP was assayedusing a Dupont RIA kit. The results are graphically presented in FIG. 6.

As shown in FIG. 6, the addition of AIT-082 increased the production ofcGMP in PC12 cells indicating that AIT-082 acts by modifying theactivity of the carbon monoxide-dependent enzyme guanylyl cyclase.

EXAMPLE 16 Effect of AIT-082 on Genetic Expression of Neurotrophin mRNA

To demonstrate that AIT-082 induced the in vivo genetic expression andresultant cellular production of neurotrophins, naturally occurring,genetically encoded molecules, as well as enhancing their activity, thefollowing experiment was performed. Induction of neurotrophin mRNA wasdetermined by northern blot analysis of astrocytes cultured withAIT-082, NGF, or both. The cells were harvested and RNA extracted at 24hours after treatment.

More particularly, astrocytes from the cerebral cortex of NIH Swiss mice(Harlan) were isolated. Briefly, newborn pups (0-24 hours) weredecapitated. Their brains were removed under aseptic conditions and wereplaced in modified Dulbecco's medium (DMEM) containing 20%heat-inactivated horse serum (Hyclone) (“complete medium”). Theneopallium was then dissected from each cerebral hemisphere and mincedinto 1 mm cubes.

The astrocytes were then isolated by mechanical dissociation. The cubeswere vortexed at maximum speed for one minute. The cell suspension wasthen passed first through 75 mm Nitex then through 10 mm Nitex. Theresulting cell suspension was diluted in complete medium to a finalconcentration of one brain per 10 ml of complete medium. Ten millilitersof the diluted cell suspension was added to each 100 mm Falcon tissueculture plate (Fisher). After 3 days the medium was replaced with 10 mlfresh complete medium and subsequently was replaced twice weekly withDMEM containing 10% heat inactivated horse serum (“growth medium”).After two weeks in culture the astrocytes formed a confluent monolayer.

For RNA extraction, astrocytes were trypsinized. The astrocytes werethen replated onto 100 mm PORN coated plates at a cell density of 10⁶cells per plate (10 ml growth medium). After 2 hrs PBS, guanosine (Guo),or GTP at 10 mM were added to the appropriate plates. Total RNA washarvested from 1.5×10⁷ cells for each treatment, 4 and 24 hrs aftertreatment using TRIzol reagent and supplier protocol (GIBCO BRL/LifeTechnologies, Inc.). For slot blots, total RNA was bound to Hybond-Nfilters (Amersham/United States Biochemicals) as described in Transferand Immobilization of Nucleic Acids and Proteins to S & S Solid Supports(S and S Protocols: Schleicher & Schuell, New Hampshire, USA). Northernblots were also performed using 10-20 mg total RNA from each sample.These were electrophoresed in 1% agarose gels containing formaldehydeand blotted onto Hybond-N filters according to S and S protocols.

The blots were probed with ³²P-labeled cDNA (NGF, NT-3 and BDNF probes)or oligonucleotide probe (FGF-2) by hybridization inPiperazine-N,N′-bis-(2-ethanesulfonic acid) (PIPES) buffer (50 mM PIPES,pH 6.8; 50 mM NaH₂PO₄; 0.1 M NaCl; 5% SDS and 1 mM EDTA) overnight at50° C. The blots were then washed twice with (2×SSC, 0.1% SDS) washbuffer at room temperature for 20 minutes each, and then with (0.1×SSC,0.1% SDS) wash buffer twice at 52° C. for 20 minutes each. 1×SSC is 0.15M NaCl and 15 mM sodium citrate, pH 7.0. Damp membranes were wrapped inSaran wrap and autoradiography was performed using Hyperfilm-MP(Amersham/USB) and a cassette with intensifying screens. Variousconcentrations (0.25 to 4 mg of total RNA), as determined byspectrophotometry, of each sample were blotted and probed so thatquantification could be done after insuring a linear film response.Quantification was performed using MCID Image Analysis (St. Catherine's,Ontario, Canada).

To provide probes, a cDNA clone of the mouse NGF gene in the plasmidpGEM.NGF(+), and cDNA clones of human NT-3 and BDNF in Bluescript wereisolated. After isolation, the cDNA probes were labeled with ³²P-dCTP(ICN Biomedicals Canada, Ltd.) using a Random Primed DNA Labeling Kit(Boehringer Mannheim Biochemica) as described in the kit.

A 40-mer antisense oligonucleotide was synthesized (MOBIX, McMasterUniversity) as a probe for FGF-2. This was complementary to the 5′ endof mouse FGF-2 coding sequence on the mRNA. The oligo was 5′ end-labeledusing polynucleotide kinase, One-Phor-All buffer, and the protocolsupplied by Pharmacia Biotech Inc., and ATPgP32 (ICN Biomedicals Canada,Ltd.).

The results of the study for the production of four differentneurotrophic factors are shown below in Table G.

TABLE G Northern Blot Analysis of Neurotrophin mRNAs from AstrocytesNeurotrophin NGF AIT-082 AIT-082 (100 mM) + mRNA Control 40 ng/ml 100 mMNGF (40 ng/ml) NGF − − ++ + FGF-2 + − ++ + BDNF + + + + NT-3 − − ++ +The conditions which produced a detectable amount of each of theneurotrophin mRNAs are indicated by a “+”, with a “++” indicating thatat least twice the detectable amount was present. Those blots which werenegative are indicated by a “−”.

The results indicate that AIT-082 induced the expression of mRNAs forseveral neurotrophic factors, including NGF. More importantly, thesedata clearly establish that AIT-082 selectively and controllably inducedthe in vivo genetic expression of at least one naturally occurringgenetically encoded molecule in a mammal treated in accordance with theteachings of the present invention. Administering this exemplary purinederivative selectively induced the expression of mRNA encoding three ofthe four identified neurotrophic factors, NGF, FGF-2, and NT-3, but didnot induce activation or derepression of the gene encoding for BDNFmRNA. This selective control coupled with the ease of administrationprovided by the compounds and methods o the present inventioneffectively overcomes the limitations of the prior art. Rather thanadministering these molecular compounds directly to cells throughcomplex and potentially dangerous techniques, the present invention isable to treat a mammalian patient utilizing traditional, noninvasivedrug delivery routes that induce the treated cells to express thegenetic material encoding the desired compounds resulting in theirdirect in vivo delivery and administration. Though potentially useful inconjunction with modified genes or other molecular biology techniques,with the present invention, genetic modification is unnecessary.

It has been shown previously that, within the hippocampus fromAlzheimer's patients, there is an altered program of gene expressionleading to aberrant levels of mRNA for neurotrophic factors. A number ofanimal and clinical studies have demonstrated that administration ofsingle neurotrophins are inadequate to treat neurodegenerative disease.Accordingly, the ability of the compounds of the present invention tostimulate the production of multiple neurotrophin mRNAs within cellssubstantially increases their potential as treatments for a variety ofneurodegenerative diseases by providing a method for the effectivedirect administration of these naturally occurring genetically encodedmolecules to a patient through the induction of their in vivo geneticexpression.

The preceding examples show that AIT-082 stimulates neuritogenesis invitro in PC12 cells alone and enhances the effect of nerve growth factor(NGF). Further, the neuritogenic effect of AIT-082 was reduced bymethemoglobin (which captures and removes nitric oxide and carbonmonoxide), methylene blue (which inhibits guanylyl cyclase), and by zincprotoporphyrin IX (an inhibitor of heme oxygenase 2, which producescarbon monoxide). The neuritogenic effect of AIT-082 was unaffected byL-NAME or NOLA, inhibitors of NO production. In addition, AIT-082stimulated the production of a number of different neurotrophic factorsas evidenced by increased mRNA levels of these factors in astrocytesafter AIT-082 administration in vitro. Moreover, since AIT-082 is orallyactive and rapidly passes the blood-brain barrier as shown in Example 2,it has significant therapeutic potential as an NGF-mimetic agent inAlzheimer's disease and in other neurodegenerative diseases.

In view of the previous results, studies were performed to demonstratethe effectiveness of using AIT-082 to treat neurodegenerative diseases.Loss of memory represents the core symptom of Alzheimer's disease as itdoes in a number of other neural afflictions. Specifically working (orepisodic) memory is impaired in Alzheimer's disease, amnesia, aging andafter hippocampal lesions in monkeys. The effects of AIT-082 inameliorating this memory loss was used to demonstrate the efficacy ofthe compounds of the present invention with respect to the treatment ofneurodegenerative diseases.

EXAMPLE 17 Comparison of Memory Trace in Different Mice Strains

The win-shift T-maze paradigm has been shown to specifically modelworking memory in rodents and is a widely accepted method. The rodent'snatural behavior is to forage for food when hungry and therefore it willnot return to the same location after it has consumed any food that waspresent. This model was not designed to account for all of the vast dataon memory. Data from hypoxia and ischemia studies, procedures whichselectively damage CA1 hippocampal cells, produce deficits in workingmemory while other types of memory are not affected. This stronglysuggest that there are several types of memories which have differentanatomical sites and most likely different neurochemical inputs.Accordingly, while the win-shift model may not account for allneurochemical inputs involved in working memory, the model does providea useful art accepted tool in designing pharmacological experiments toprovide information on the mechanism by which memory can be modified.

Male Swiss Webster mice six months (young adult) and eleven months (old)of age, obtained from the National Institute on Aging, were maintainedin individual cages, on a 22 hour light/dark cycle with continuousaccess to water. Food was limited so that the mice stabilized at 80% offree feeding weight. Mice were weighed and handled daily for one week.The win-shift model was run as described in the literature and consistsof a T-maze in which the correct response alternates after each correcttrial. The interval between trials is varied and allows for thedetermination of the longest period between trials that a subject canremember the correct response on the previous trial. This allows themeasure of the duration of the memory trace. A score of 5 (5 correctresponses per 10 trials, 50% correct) is considered chance; that is, theanimal does not remember which box it selected for positive reward onthe previous trial. The reward goal box is alternated after each correcttrial. Ten trials per mouse are run each day. If the animal establishesa spatial learning set (right side only), they would return to the samegoal each trial and have a correct response rate of significantly lessthan 50% correct. The latency time to leave the start box is recorded asa measure of motivation, the running time (the time from leaving thestart box to reaching the goal box) is recorded as a measure ofperformance, and the number of correct responses as a measure of memory.

The data in Table H illustrate the effect of increasing the inter-trialinterval in young adult mice without any drug treatment.

TABLE H Effect of Inter-Trial Interval in Win-Shift Paradigm⁽¹⁾Inter-Trial Interval (Seconds) 30 60 90 120 150 Swiss Webster mice 7.5*7.5* 5.0  C57BL/6 mice 7.0* 7.4* 7.0* 7.8 5.6 ⁽¹⁾Score is the meannumber of correct responses per 10 trials. Saline was administered 1hour prior to testing. *p < 0.05. Data analysis following significantANOVA, a Dunnett test was run comparing drug tested groups withcontrols. An Arc Sign transformation was performed on percentage data.

From the data in Table H, it can be seen that Swiss Webster mice arecapable of remembering the win-shift strategy when the inter-trial delayinterval is 30 or 60 seconds. Few mice with saline treatment scoredabove chance (50%) with the 90-second inter-trial delay interval. Thesedata indicate that the “memory trace” in these animals disappearsbetween 60 and 90 seconds All drug evaluation tests in normal adultSwiss Webster mice were conducted with the 90-second inter-trialinterval except where indicated otherwise. In C57BL/6 mice, the durationof the memory trace was 120 seconds.

EXAMPLE 18 Effect of AIT-082 on Memory Trace Duration

The activity of AIT-082 was compared with tacrine (THA) andphysostigmine (PHY), experimental anticholinesterase agents whichenhance memory in animals. The drugs were also evaluated for theireffects on locomotor activity. In the win-shift memory paradigm, AIT-082was evaluated for its ability to induce tolerance after 18 days of drugadministration. In addition AIT-082 was tested for its activity tomodify learning in a modified T-maze discrimination task.

The drugs used in this example are4-((3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl)amino) benzoic acid(AIT-082), as an exemplary potassium salt, tacrine hydrochloride(tetrahydroaminoacridine, THA, Sigma Chemical Co., St. Louis, Mo.), andphysostigmine, hemisulfate salt (PHY, Sigma Chemical Co., St. Louis,Mo.). The drugs were dissolved in saline and prepared fresh daily. Allinjections were made at a volume of 0.1 ml/10 grams body weight. Whentesting drug effects, intraperitoneal (i.p.) injections of AIT-082 orTHA were made one hour prior to the start of testing. Due to its shorterduration of action, PHY was injected 30 minutes prior to testing.Control subjects receive a similar injection of saline (vehicle).

To determine the duration of the memory trace, subjects wereadministered drug or saline and 30 minutes (PHY) or 1 hour (AIT-082 orTHA) later they were given a single reference run with the milk rewardin both goal boxes. After the indicated inter-trial delay, subjects werereturned to the start box and given the first test trial with the milkreward only in the goal box opposite to the one entered on the previouscorrect trial. The subjects were given 10 trials with the rewardalternating only after correct responses.

To determine if tolerance to the biological effects of AIT-082developed, drug or saline was administered daily for 18 days prior tothe testing in the standard win-shift paradigm.

Subjects were also trained in the same T-maze used for the win-shiftmodel discussed above. As in the win-shift method, subjects were shapedand then given a single reference run in which reward was available inboth goal boxes. The subject was only allowed to consume the milk rewardin the goal box selected. On the next run, the reward and thus thecorrect response was in the same goal box selected for the reference runand was not alternated. The subject was required to learn that there wasno shift in the goal box for the correct response. The subjects weregiven 10 trials per day and continued until the subject had at least 8out of 10 correct responses on two consecutive days. The number of daysto reach this criteria of performance was recorded. After the subjectreached criteria, the goal box for the correct response was reversed.The number of days taken to reach criteria on reversal was recorded.

The results of the T-maze learning task and win-shift memory test arepresented in Table I directly below.

TABLE I Effect of AIT-082, THA and PHY at 90-Second Inter-Trial Intervalin Swiss Webster Mice THA AIT-082 PHY Control Dosage (mg/kg) Type ofTest⁽¹⁾ Saline 1.25 0.5 30.0 0.125 Win-shift Memory Test Correctresponses 4.6  7.1* 6.5* 8.2* 6.5 (Correct responses/10 trials) Latencytime (seconds) 2.68  8.22* 1.95 2.03 Running time (seconds) 1.95  3.65*2.20 1.95 2.65 Locomotor Activity⁽²⁾ 343 671* 323 378 N/T T-mazeLearning (days to reach criteria) Learning 3.6 N/T³ 3.0 3.3 N/T Reversal4.2 N/T 3.78 3.5 N/T Tolerance 4.9 N/T N/T 7.6* N/T (Correctresponses/10 trials) ⁽¹⁾at least 8 animals were run per group.⁽²⁾Spontaneous movements per hour. ⁽³⁾Not tested. *Indicates p < 0.05.Data analysis following significant ANOVA, a Dunnett test was runcomparing drug tested groups with controls. An Arc Sine transformationwas performed on percentage data and latencies were transformed toreciprocal time scores or speed scores.

As can be seen from the data in Table I, AIT-082 treatment resulted inan increased number of correct responses (memory) compared to salinecontrol. While the effect was in the same range as with THA and PHY,both THA and PHY also increased latency time (prolonged the time toleave the start box, evidencing decreased motivation) and THA increasedspontaneous locomotor activity. AIT-082 had no effect on learning orreversal and no tolerance developed to the memory enhancing effect ofAIT-082 after 18 days of pre-treatment. Only AIT-082 enhanced memoryfunction without affecting learning, motivation, performance andlocomotor activity. Similar data have been observed with oraladministration of AIT-082.

EXAMPLE 19 Effect of AIT-082 Dosage on Memory Trace Duration

The dose response and duration of action of AIT-082 was studied in youngadult Swiss Webster mice. The results are presented as the percentcorrect response over chance; chance being 50% correct. As shown in FIG.7, AIT-082 is active in improving memory in normal adult Swiss Webstermice over a dose range from 0.5 to 60 mg/kg, with the optimal effect at20 to 30 mg/kg. Further, as shown in FIG. 8, the onset of action israpid (1 hour, data not shown) and lasts for more than seven days aftera single dose of 60 mg/kg. Those skilled in the art will appreciate thatthe extended duration of the drug's effects will substantially lower thefrequency of administration providing benefits in terms of patientcompliance and cost.

EXAMPLE 20 Effect of AIT-082 on Memory Trace Duration in C57BL/6 Mice

Previous work has established that normal adult Swiss Webster mice havea memory trace duration of 60 seconds in the win-shift paradigm whichmay be increased by the administration of AIT-082. In order to furtherdemonstrate the applicability and operability of the methods andcompositions of the present invention, an alternative strain of micehaving a different duration of memory trace was administered AIT-082,using the preceding protocol. The results are shown in Table J directlybelow.

TABLE J Duration of Memory Trace in C57BL/6 Mice Treatment GroupsControl AIT-082 Physostigmine (Saline) (30 mg/kg) (0.125 mg/kg)Inter-trial No. above No. above No. above interval chance/ chance/chance/ (sec) Total # Correctà Total^(#) Correct⋄ Total^(#) Correctà  303/5 70 ± 11**  60 3/5 70 ± 16**  90 4/5 70 ± 6** 120 4/5 78 ± 16** 1501/5 56 ± 10 180 2/7 58 ± 12 4/6 70 ± 15** 3/6 65 ± 16* 210 4/6 78 ± 15**1/6 53 ± 9 240 0/6 50 ± 6 270 0/6 50 ± 6 # = No. subjects above chance(60% correct)/Total No. subjects tested à = Mean score ± S.E. ** = p <0.01 (t-test against chance) * = p < 0.05 (t-test against chance)

Typically, in the win-shift foraging paradigm, C57BL/6 mice have aduration of memory trace of 120 seconds. As shown in Table J, at 30mg/kg i.p., AIT-082 prolonged the duration of the memory trace to over210 seconds. While physostigmine also prolonged the duration of thememory trace from 120 to 180 seconds in this model, it was not as activeas AIT-082.

EXAMPLE 21 Treatment of Age Induced Memory Disorders using AIT-082

In light of the preceding results, studies were performed to demonstratethat AIT-082 improves memory in mammals with neuronal disorders as wellas in healthy subjects. Twelve-month old male Swiss Webster mice werescreened for performance in the win-shift foraging test. Subjects weretested at various time delays, beginning at 10 seconds and increasingthe inter-trial time interval to 30, 60, 90 and 120 seconds. The resultsfor untreated mice are shown in Table K directly below.

TABLE K Age-Induced Working Memory Deficits in Swiss Webster MiceDuration of No. of % of Degree of Memory Memory Trace Subjects SubjectsImpairment less than  6 25 Severe 10 seconds 10 seconds  8 33 Moderate30 seconds 10 42 Mild Total 24 100 

The results in Table K demonstrate that individual subjects can beclassified by the degree of working memory impairment. Subjects withsevere impairment could not remember the correct response at 10 secondswhile subjects with mild deficit could remember the correct responsewith a 30 second inter-trial interval but not at 60 seconds. Subjectswith a moderate deficit could remember the correct response with a 10second inter-trial interval but not 20 at 30 seconds. Thus, thewin-shift model can detect age-induced impairments in working memory. Aswill be appreciated by those skilled in the art, this observation isimportant since it provides the ability to use age-matched subjects withvarying degrees of impairment for evaluation of potential therapeuticagents.

Following the establishment of a baseline, six subjects in each of thethree groups were treated with AIT-82 (30 mg/kg, one hour beforetesting) or physostigmine (0.125 mg/kg, 30 minutes before testing) usingthe win-shift foraging test. The results are presented in Table Ldirectly below and graphically represented in FIG. 9.

TABLE L Effect of AIT-082 and PHY on the Duration of Memory Trace inSwiss Webster Mice with Age-Induced Deficits Degree Inter-trial ofInterval Control AIT-082 PHY Deficit (sec) (Saline) 30 mg/kg 0.125 mg/kgMild 60 0/6  6/6*  5/6* 90 4/6 3/6 120 2/6 2/6 150 1/6 2/6 180 1/6 1/6210 0/6 0/6 Moderate 30 0/6  4/6* 1/6 60 2/6 0/6 90 0/6 Severe <10 0/60/6 0/6 Data is presented as the number of subjects performingsignificantly above chance/total number of subjects; *Indicates p < 0.05(t-test)

Six subjects had a severe deficit with no memory trace, they could notremember the task at 10 seconds. None of these subjects showed memoryrestoration with either AIT-082 or PHY treatment. In the six subjectswith a moderate memory deficit who had a duration of memory trace of 10seconds, AIT-082 increased the duration of the memory trace to greaterthan 30 seconds in 4 subjects (67% of the subjects) and increased thememory trace to greater than 60 seconds in two subjects (50%). In thesix subjects with a mild memory deficit who had a duration of memorytrace of 30 seconds, AIT-082 increased the duration of the memory tracein 2 subjects to 60 seconds, in 2 subjects to 90 seconds and in onesubject each to 120 and 180 seconds. PHY increased the duration of thememory trace from 10 seconds to 30 seconds in only one animal in themoderate deficit group. In the mild deficit group, PHY increased theduration of the memory trace in 2 subjects to 60 seconds, in one subjectto 90 seconds and in two subjects to at least 180 seconds. Thus, AIT-082is more active than physostigmine in the moderate deficit group and atleast as active in the mild deficit group.

EXAMPLE 22 Treatment of Age Deficit Memory Disorders using AIT-082

Twelve-month old male C57BL/6 mice were screened for performance in thewin-shift foraging test. Subjects were tested at various inter-trialtime intervals. Subjects who could not perform to criteria (>60%correct) at 10 seconds delay were classified as having a severe deficit.Subjects who performed to criteria at 10 seconds but not at 30 secondswere classified as having a moderate degree of deficit and subjects whoperformed to criteria at 30 seconds but not at 60 seconds wereclassified as having mild deficit. As in Example 21, subjects in eachgroup were treated with either AIT-082 or PHY to determine the extent towhich the working memory trace was prolonged. The results are presentedin Table M directly below and graphically represented in FIG. 10.

TABLE M Effect of AIT-082 and PHY on the Duration of Memory Trace inC57BL/6 Mice with Age-Induced Deficits Degree Inter-trial of IntervalControl AIT-082 PHY Deficit (sec) (Saline) 30 mg/kg 0.125 mg/kg Mild 600/6  4/4*  7/8* 90  2/4* 3/8 120 2/8 150 2/8 180 2/8 210 0/8 Moderate 106/6  6/6* 6/6 30 0/6 4/6 1/6 60 1/6 0/6 90 0/6 Severe <10 0/6 0/6 0/6Data is presented as the number of subjects performing significantlyabove chance/total number of subjects; *Indicates p < 0.05 (t-test)

In the mild deficit group, AIT-082 prolonged the duration of the memorytrace from 30 to 90 seconds, from 10 to 30 seconds in the moderatedeficit group. While PHY prolonged memory in the mild group, it wasineffective in the moderate group. Therefore AIT-082 restored workingmemory deficits in both normal mice and mice with age induced neronaldisorder for both Swiss Webster and C57BL/6 strains. Specifically, theresults show the AIT-082 restores working memory in mice with mild andmoderate memory deficits. Based on the other Examples previouslyprovided it is reasonable to conclude that it accomplishes thisrestoration by modifying the carbon monoxide dependent guanylyl cyclasesystem.

EXAMPLE 23 Prophylaxis of Age Deficit Memory Disorders using AIT-082

It has been observed that age-induced memory deficits typically begin tomanifest themselves in mice between 14 and 16 months of age. Therefore,the treatment of mice was begun at 14 months of age with AIT-082 (30mg/kg/day) in their drinking water. The animals were measured monthlyfor their memory using the win-shift foraging tests previouslydescribed. The results are shown in FIG. 11 and show that theadministration of AIT-082 delayed the onset and severity of memorydeficits.

EXAMPLE 24 Prophylaxis of Alcohol-Induced Deficit Memory Disorders usingAIT-82

In order to demonstrate the broad applicability of the methods of thepresent invention with respect to different types of neurodegenerativedisorders, AIT-082 was used to retard the production of alcohol inducedmemory deficit. Six month old male C57BL/6 mice were evaluated in thewin-shift model in combination with treatment with ethanol, anon-specific memory suppressant, and AIT-082. Subjects were treated withsaline (control) or AIT-082 (30 mg/kg. i.p.) 1 hour prior to testing.Ethanol was administered at a dose of 0.5 gm/kg i.p. ten minutes priorto testing. The results of a pilot study are presented in Table Ndirectly below.

TABLE N Working Memory Deficit Produced by Ethanol and its Reversal byAIT-082 Treatment Ethanol + Control Ethanol AIT-082 Correct trials^(1,2)8.08 ± 0.29  6.5 ± 26* 7.89 ± 0.54† Latency time (sec)² 1.24 ± 0.17 1.18± 0.10 1.77 ± 0.27 Running time (sec)² 1.44 ± 0.35 1.17 ± 0.08 3.22 ±0.61*† Number of subjects 13 13 9 ¹Indicates mean number of correctresponses per 10 trials; ²Indicates mean values ± S.E.; *Indicates p <0.05 (t-test) compared to control; † lndicates p < 0.05 (t-test)compared to ethanol.

The results in Table N demonstrate that it is possible to identify adose of a blocking agent that can produce a memory deficit as measuredin the win-shift model. Ethanol was selected as a non-specific blockingagent and its effects were reversed by administration of AIT-082 priorto the treatment with ethanol. Therefore it would appear feasible toevaluate other more specific blocking agents which have activity atspecific receptor sites.

In addition to AIT-082 other purine derivatives are believed to play arole in neuronal survival, synaptogenesis and recovery of functionfollowing injury or cell death in the central nervous system. Forexample, similarities between guanosine and AIT-082 indicate thatAIT-082 and guanosine act through comparable mechanisms. That is, bothappear to act as carbon monoxide dependent guanylyl cyclase modulators.Further, it is known that after cells are damaged, they leak massiveamounts of both purine nucleosides and nucleotides to the extracellularspace. The extracellular concentration of guanosine in the region of afocal brain injury may reach 50 mM and is elevated up to five fold forat least seven days. Therefore, following injury, astrocytes or glia andneurons are exposed to high extracellular concentrations of guanosine.

Accordingly, the following studies were undertaken in order todemonstrate the effectiveness of using other exemplary purinederivatives such as guanosine to modulate the carbon monoxide dependentguanylyl cyclase system.

EXAMPLE 25 Astrocytes Produce Trophic Gactors Upon Exposure to Guanosineand GTP

Astrocytes appear to proliferate in response to extracellular guanosineor guanosine triphosphate (GTP). GTP or guanosine may also stimulate therelease of trophic factors from cultures of neocortical astrocytes fromneonatal mouse brains. Astrocytes were incubated with differentconcentrations of guanosine or GTP respectively. Neurotrophinimunoreactivity in the culture medium from treated cells was thenmeasured by ELISA.

Briefly, 96 well Falcon plates (Fisher) were coated with 1 mg/ml ofsheep mono-specific anti-NGF IgG (affinity column purified) contained in0.1M sodium carbonate buffer pH 9.6. After an overnight incubation at 4°C. blocking solution (PBS with 10% goat serum) was added to removeexcess antibody. After a four hour incubation at room temperature theplates were washed three times with PBS containing 0.05% Tween 20. Theconditioned media and standard 2.5S HPLC purified NGF were added andincubated overnight. The next day plates were washed 3 times withPBS-0.05% Tween 20. The secondary antibody, rabbit mono-specificanti-NGF IgG conjugated with β-galactosidase (Pierce-SPDP method) (1:500dilution) was added. The plates were incubated overnight at 4° C. Thenext day the plates were washed 3 times with PBS-0.05% Tween 20. Toeach, well substrate, 0.2 mM 4-methylumbelliferyl-β-galactoside (MUG) in0.1 M phosphate buffer (1 MM MgCl₂ pH 7.2) was added. After a 4 hourincubation at room temperature the reaction was stopped by the additionof 0.1 M glycine, pH 10.3. Samples were then read using Microfluor ELISAreader (excitation 360 nm; emission 450 nm). The sensitivity of thisassay was 10 pg/well NGF.

The ELISA assay detected neurotrophins NGF and NT-3 with ahnost equalaffinity and BDNF with 100 times less affinity. As shown in FIGS. 12Aand 12B, both guanosine and GTP increased the amount of NGF-likeimmunoreactivity in the culture medium. The astrocytes exposed to thevarious levels of guanosine produced a much stronger response than thoseexposed to equivalent concentrations of GTP.

EXAMPLE 26 Astrocytes Produce Neurotrophic Factors Upon Exposure toGuanosine

In order to confirm the results of the previous assay, mRNA levels ofthe tropic factors FGF-2 and NGF were measured in astrocytes which hadbeen exposed to guanosine. The mRNA levels were measured using the sameprotocol used previously in Example 16. As shown in FIGS. 13A and 13B,the addition of guanosine increased NGF and FGF-2 mRNA at 4 hours and at24 hours, respectively, after it was added to astrocytes. The observedincrease in neurotrophin mRNA is important following brain injury orrecovery from brain injury when the extracellular concentration ofguanosine is considerably high. As cells are exposed to a highconcentration of guanosine for several days following brain injury, thisdata indicates that guanosine may be responsible for some of therecovery of function.

As previously discussed, an agent that can penetrate the blood brainbarrier and increase concentrations of neurotrophic factors as measuredhere by mRNA levels should have a substantial positive effect onneuronal survival and on the formation of collateral nerve circuits. Inturn, this should enhance functional recovery in many differentneurological diseases or after damage to the nervous system.

EXAMPLE 27 Neurons Undergo Neuritogenesis Upon Exposure to Guanosine

In addition to changes in glia or astrocytes, important neuronal changesalso take place following focal brain injury. Neuritic processes ofsurviving neurons may undergo neuritogenesis. Accordingly, based onprevious results using AIT-082, studies were performed to demonstratethat guanosine may also modify carbon monoxide guanylyl cyclase tostimulate neuritogenesis. As previously discussed, because PC12 cellsconstitute a homogeneous population of neuronal-like cells, withoutcontaminating astroglia-type cells, the direct effects of the exemplarypurine derivatives of the present invention on neurite outgrowth inthese cells can be observed easily. Accordingly, PC12 cells were exposedto guanosine and adenosine with and without NGF and monitored as inExample 12. The effects of exposure to purine derivatives in thepresence of NGF are shown in FIG. 14A while exposure to purinederivatives in the absence of NGF is shown in FIG. 14B. A directcomparison of the effects of these purine derivatives in the presence orabsence of NGF is shown for each compound in FIG. 14C.

As shown in FIG. 14A, guanosine, but not adenosine, enhanced the neuriteoutgrowth induced by NGF in PC12 cells after 48 hours. The enhancementwas significant over that of NGF alone at guanosine concentrations of 30and 300 mM. Adenosine did not enhance NGF induced neurite outgrowth atany concentration. This indicates that neurite outgrowth induced bypurines is not just a generalized phenomenon.5′-N-ethylcarboxamidoadenosine (NECA), an adenosine A₁ and A₂ receptoragonist, also enhanced neuritogenesis, but not to the same extent asguanosine.

On their own, in the absence of NGF, both adenosine and guanosineslightly increased the proportion of cells with neurites as shown inFIG. 14B. The effects of guanosine at both 30 and 300 mM were greaterthan adenosine at the same concentrations. In the presence of NECA,there was little stimulation of neurite outgrowth. Because the effectsof the compounds in the presence of NGF were much more readily scoredand less variable from experiment to experiment than with the compoundsalone, most of the data for enhancement of neurite outgrowth wasdetermined in the presence of NGF.

The comparative data shown in FIGS. 14A and 14B and emphasized in FIG.14C show that guanosine causes some neurite extension, but can alsoreact synergistically to enhance the trophic effects of NGF. Adenosine,although slightly enhancing neurite outgrowth on its own does notenhance the effects of NGF. Interestingly, NECA but not adenosine couldsynergistically enhance the actions of guanosine, both in the presenceand absence of NGF as shown in FIG. 14C. The fact that adenosine did notincrease NGF-dependent neurite outgrowth in PC12 cells but thatguanosine did, suggests that they interact differently with PC12 cells.Adenosine would interact with adenosine receptors, such as the A2purinoceptor. This would activate adenylate cyclase which increasesintracellular cAMP. NECA apparently acts in this manner. But the effectsof NECA were synergistic with those of guanosine. This indicates thatguanosine and NECA use different signaling pathways to enhance neuriteoutgrowth.

EXAMPLE 28 Various Purine Derivatives Provide Different Rates ofNeuritogenesis

In view of the previous results, other exemplary purine derivatives wereexamined to demonstrate the specificity of those compounds whichmodulate carbon monoxide dependent guanylyl cyclase to modify neuralactivity. Specifically, different concentrations of the purinederivatives inosine, hypoxanthine and xanthine were tested in thepresence of NGF using the protocol of Example 12 to demonstrate theirability to modify neural activity.

As shown in FIG. 15A, inosine only slightly enhanced neurite outgrowthover that produced in cells treated with NGF alone. This was true forconcentrations of inosine ranging from 0.3 to 300 mM. FIG. 15A alsoshows that the action of inosine on the enhancement of neurite outgrowthwas much less effective than that of guanosine.

FIGS. 15B and 15C also show that hypoxanthine and xanthine each producedresults similar to that of inosine on NGF-induced neuritogenesis. InFIG. 15C xanthine, in concentrations from 0.3 to 30 mM (300 mM was toxicto the cells), only slightly enhanced NGF-induced neurite outgrowth.FIG. 15B shows that hypoxanthine showed the greatest, although stillmodest, enhancement at concentrations of 0.3 and 300 mM, although otherconcentrations had no significant enhancement. Even though someenhancement of neurite outgrowth was observed with hypoxanthine, therelative amount of enhancement was not nearly as great as was the effectof guanosine. These results indicate that inosine, xanthine andhypoxanthine do not modulate the carbon monoxide-dependent guanylylcyclase system to modify neural activity but rather influence othersignaling mechanisms to the extent that enhancement was observed.

EXAMPLE 29 Effects of AIT-34 on Neuritogenesis

To demonstrate the effects of compounds similar to AIT-082 onneuritogenesis, PC12 cells were exposed to AIT-34, otherwise known as3-(1,6 dihydro-6-oxo-9h purin-9-yl)-N-(3-(2-oxopyrrolidin-1-yl)propyl)propanamide, during growth and monitored according to Example 12. Asshown in FIG. 16, different concentrations of AIT-034 did not enhanceNGF-induced neuritogenesis as is observed with AIT-082.

EXAMPLE 30 Effects of ATP and GTP on Neuritogenesis

To further demonstrate that purine derivatives having differentfunctional groups may be used in accordance with the teachings of thepresent invention, PC12 cells were exposed to adenosine triphosphate(ATP) and guanosine triphosphate (GTP) and monitored for neuritogenesisusing the protocol of Example 12.

In a manner very similar to the actions of adenosine and guanosine onneurite outgrowth in PC12 cells, their corresponding nucleotides ATP andGTP had parallel effects on neurite outgrowth. As shown in FIG. 17, ATPdid not enhance neuritogenesis in either NGF treated cells or on itsown. In sharp contrast, GTP at 30 and 300 mM did enhance neuritogenesisin the presence of NGF and further elicited neurite outgrowth on itsown.

However, as shown in FIG. 18, GTP) did not appear to be acting as asource from which guanosine was released in a controlled manner. If GTPwere hydrolyzed to guanosine diphosphate (GDP), guanosine monophosphate(GMP) and finally to guanosine by ectoenzymes, one would predict thatGDP and GMP would also enhance neurite outgrowth from PC12 cells becausethose less highly phosphorylated molecules would also be converted toguanosine by hydrolysis. Yet, neither GDP nor GMP were effective aloneor with NGF in eliciting neurite outgrowth. By way of comparison, theadenine-based compounds all had an inhibitory effect.

EXAMPLE 31 Guanosine but not GTP Increases cGMP in PC12 Cells

Based on the previous examples, a study was conducted to demonstrate theneuritogenic mechanisms of GTP and guanosine respectively. Guanosine andGTP have been shown to increase intracellular cyclic 3′,5′-guanosinemonophosphate (cGMP) in arterial smooth muscle. Since cGMP analogueshave been reported to stimulate neurite outgrowth from neuroblastomacells it was possible that both guanosine and GTP might exert theireffects through increasing intracellular cGMP. As shown in FIG. 19,guanosine increased intracellular cGMP in PC12 cells as determined byradioimmunoassay using the protocol detailed in Example 15. Such anincrease would be expected of a carbon monoxide dependent guanylylcyclase modulator. In contrast, it was found that GTP did not increaselevels of cGMP, indicating that any GTP-stimulated neuritogenesis occursby another mechanism.

EXAMPLE 32 Use of Non-Selective Inhibitors of Guanylyl Cyclase ReducesGuanosine Neuritogenesis

To demonstrate that guanosine modifies the carbon monoxide-dependentguanylyl cyclase system, studies were conducted to show that increasedlevels of intracellular cGMP were necessary for guanosine to enhanceNGF's neuritogenic effects on PC12 cells. In particular, differentconcentrations of three inhibitors of guanylyl cyclase were added to PC12 cells with guanosine. Neuritogenesis was then determined using theprotocol of Example 12.

Methylene Blue (MB) inhibits soluble guanylyl cyclase (sGC), the enzymethat synthesizes cGMP. As shown in FIG. 20A the addition of MB (0.1-5mM) to cultures of PC12 cells abolished the synergistic effects ofguanosine with NGF. Conversely, MB had no effect on NGF-stimulatedneurite outgrowth.

LY83583 inhibits both particulate and sGC. FIG. 20B shows that theneurite outgrowth response elicited by guanosine was inhibited byLY83583, but the response elicited by NGF was unaffected. The mechanismby which LY83583 inhibits guanylyl cyclase is unresolved, but is likelyindirect, involving glutathione metabolism. Therefore, two non-selectiveinhibitors of guanylyl cyclase, each with a different mechanism ofaction, attenuated the neuritogenic action of guanosine.

These data indicate that guanosine and NGF act through differentmechanisms. They also indicate that increases in intracellular cGMP werenecessary, although possibly not sufficient, for guanosine to exert itsneuritogenic effects.

To test whether increases in cGMP were sufficient to cause neuriteoutgrowth, atrial natriuretic factor (ANF) was added to cell cultures ina manner similar to that used for guanosine. ANF is a hormone whose onlyknown signal transduction pathway is through activation of particulateguanylyl cyclase. As shown in FIG. 20C, ANF, like guanosine, enhancedNGF-stimulated neurite outgrowth from PC12 cells indicating thatincreased intracellular cGMP production, induced by carbon monoxidedependent guanylyl cyclase or other mechanisms, assisted in stimulatingneurite outgrowth.

EXAMPLE 33 Nitric Oxide or Carbon Monoxide Promotes GuanosineNeuritogenesis

Since guanosine increased intracellular cGMP as shown in Example 31,studies were performed to demonstrate whether its signal could betransduced through production of NO or CO. If NO were involved, thenaddition of nitric oxide donors that liberate NO should mimic theeffects of guanosine.

PC12 cells were grown for 48 hours in the presence of sodiumnitroprusside (SNP) or sodium nitrite (SN), both of which liberate NO.Alone, neither SNP nor SN elicited neurite outgrowth from PC 12 cells.However, like guanosine, both SNP and SN enhanced NGF-mediated neuriteoutgrowth in a synergistic manner as shown for the addition of SN inFIG. 21. Further confirming the effect, FIGS. 22A and 22B show that theneuritogenic properties of the NO donors were inhibited by bothhemoglobin (Hb) and methemoglobin (MB). Both are substances whichscavenge NO and CO with high affinity and preclude these agents frombeing used as signal transmitters.

Accordingly, if NO or CO mediates the neuritogenic effects of guanosine,then these effects should be reduced by addition of hemoglobin to thecultures. The expected effect is clearly shown in FIG. 23 where Hb(0.1-1 mM) inhibited the neuritogenic effects of guanosine but not thoseof NGF. This indicates that the neuritogenic action of guanosine, butnot that of NGF, requires synthesis of NO or CO.

Several facts indicate that it is CO rather than NO which interacts withguanosine to modify neural activity. For example, if the effects ofguanosine were mediated through NO, then addition of guanosine to thePC12 cells should stimulate cNOS in PC12 cells to produce NO. However,cNOS had not been reported in PC12 cells and untreated (guanosine andNGF naive) PC12 cells did not stain for diaphorase, an enzyme thatco-localizes with NOS. Since cNOS is calcium/calmodulin-sensitive, itsactivity should increase after adding a calcium ionophore, thus leadingto increased cGMP levels. Addition of the ionophore A23187 to culturesof PC 12 cells failed to elicit an increase in cGMP.

EXAMPLE 34 Carbon Monoxide, not Nitric Oxide, Mediates the Effects ofGuanosine on Neuritogenesis

Based on the results of the previous examples, studies were performed todemonstrate that the purine derivatives of the present invention,including guanosine, modulate the carbon monoxide-dependent guanylylcyclase system to modify neural activities.

As in Example 6 where it was shown that carbon monoxide mediates theeffects of AIT-082 through the use of inhibitors, the same techniquesdemonstrate that guanosine also interacts with the carbon monoxidedependent system. Specifically, as shown in FIG. 24, the cNOS inhibitorL-nitro arginine methyl ester (L-NAME) did not affect the ability ofguanosine to enhance NGF-mediated neurite outgrowth. These data confirmthat cNOS was not involved in the signal transduction pathway thatmediated the neuritogenic effects of guanosine on PC 12 cells.

To further demonstrate that CO, rather than NO, mediated theneuritogenic effects of guanosine, zinc protoporphyrin IX (ZnPP), whichinhibits heme oxygenase and hence inhibits CO synthesis, was added tothe cells during growth. As shown in FIG. 25, ZnPP abolished theneuritogenic effects of guanosine, but did not affect those of NGF. Incontrast, a related protoporphyrin derivative, copper protoporphyrin IX(CuPP), does not inhibit heme oxygenase. Accordingly, FIG. 26 shows thatcopper protoporphyrin IX did not reduce the ability of guanosine toenhance NGF-dependent neurite outgrowth from PC12 cells. As withAIT-082, these data indicated that guanosine increased CO synthesis. Inturn, CO activated sGC and increased intracellular GMP, therebypromoting neuritogenesis.

EXAMPLE 35 Inosine Pranobex Enhances Neuritogenesis

To provide further evidence of the scope and operability of the presentinvention, neuritogenic studies were performed using inosine pranobex.Specifically, inosine pranobex is a mixture of inosine and DIP-PacBa ata 1:3 molar ratio. Various concentrations of this compound were added toPC12 cells with NGF which were then monitored according to the protocolof Example 12.

As shown in FIG. 27, inosine pranobex substantially enhanced the amountof neurite outgrowth of the treated cells. The curve shown in FIG. 27represents the different levels of inosine pranobex plus saturatingconcentrations of NGF while the horizontal lines represent the NGFcontrol with attendant confidence levels. Here the treated cells areabove the control baseline at most of the selected concentrations.

The modification of neural activity in accordance with the teachings ofthe present invention may be used to treat neurodegenerative diseases inorder to provide recovery of neural function. Thus the methods of thepresent invention may be used to treat neurodegeneration from any causeincluding disease, trauma, age and exposure to harmful physical orchemical agents. Similarly, the methods disclosed herein may be used totreat neurological diseases including, but not limited to, Alzheimer'sDisease and related degenerative disorders, Parkinson's disease andrelated disorders such as striato-nigral degeneration, spino-cerebellaratrophies, motor neuronopathies or “motor system diseases” includingamyotrophic lateral sclerosis, Werdnig-Hoffman disease,Wohlfart-Kugelberg-Welander syndrome and hereditary spastic diplegia,damage to neurons by ischemia (as in strokes), anoxia, or hypoglycemia(as, for example after prolonged circulatory arrest), Huntington'sdisease, cerebral palsy, multiple sclerosis, psychiatric disordersincluding affective disorders, schizophrenia, epilepsy and seizures,peripheral neuropathies from any cause, learning disabilities anddisorders of memory. Also, damage to neurons or their processes byphysical agents such as radiation or electrical currents or by chemicalagents including alcohol, aluminum, heavy metals, industrial toxins,natural toxins and legal or illegal drugs may be treated. The methodsmay further be used to treat victims of trauma to the brain or spinalcord resulting in neuronal damage or age related conditions such asbenign forgetfulness and deterioration of sensory, motor, reflex orcognitive abilities due to loss of neurons or neuronal connectivity.Simply administering an effective dosage of the carbon monoxidedependent guanylyl cyclase modulating purine derivative to a subjectsuffering from any of the foregoing neural disorders will induceintracellular neuronal changes producing restoration of function.

Specifically, modification of the carbon monoxide dependent guanylylcyclase system in accordance with the methods of the present inventionproduces changes in neural activity in neurons and glia cells includingastrocytes. For example, using the present invention the neural activityof astrocytes may be modified to synthesize various neurotrophic factorsand cytokines including fibroblast growth factor (FGF), nerve growthfactor (NGF), brain derived neurotrophic factor (BDNF) andneurotrophin-3 (NT-3). These factors can influence the sprouting ofneuritic processes from surviving neurons as well as promote thedevelopment of new cells. New synapses may then form and provide somerecovery of function. These neurotrophic factors also play aneuroprotective role, thus inducing their production can amelioratefurther neural damage.

Numerous purine derivatives may be used in accordance with the teachingsof the present invention. However, the ability to modify neural activityby modulating the carbon monoxide dependent guanylyl cyclase system isnot a general property of all purines or purine derivatives. Forexample, as shown in the data below, inosine, adenosine, hypoxanthineand xanthine were all relatively ineffective at modifying neuralactivity. Other purine derivatives which failed to modify neuralactivity include 3-(6-amino-9H-purin-9-yl)propionic acid, ethyl ester(AIT-0026),3-(1,6-dihydro-6-oxo-9H-purin-9-yl)-N-{3-(2-oxopyrolidin-1-yl)propyl)propanamide(AIT-0034) and propentofylline. Moreover, while other purines and purinederivatives such as 5′-N-ethylcarboxamidoadenosine (NECA) were shown tostimulate neurite outgrowth, they did not do so by modulation of thecarbon monoxide dependent guanylyl cyclase mechanism. Accordingly, thescope of the invention is defined by the functional reactivity of purinederivatives which modify neural activity as described herein and asshown by the data presented. Of course, those skilled in the art willappreciate that functionally equivalent isomers, analogues andhomologues of the compounds of the present invention may be substitutedto provide the desired neural modifications.

EXAMPLE 36 AIT-082 Protects Against Neurotoxicity in vitro and in vivo

Enhanced release of glutamate has been implicated in the pathophysiologyof neuronal degeneration following acute disorders of the brain.Specific antagonists of glutamate receptors are not therapeuticallyuseful since they impair synaptic plasticity and excitatorytransmission, thus reducing learning and memory.

High levels of extracellular adenosine have been found in thesepathological conditions and this overproduction presents an opportunityto reduce the neurodegeneration since extracellular adenosine inhibitsglutamate release. However, adenosine A1 receptor agonists have not yetbeen introduced in current therapy because of their many side effectsand unsuitable clinical pharmacokinetics.

Glial cells are a major source of extracellular purines andneuroprotective neurotrophic factors (NFs) in the CNS. Systemicadministration of NFs as therapeutic agents is limited by their poorpenetration across the blood-brain barrier and by the occurrence ofperipheral side effects. Thus, good therapeutic strategy for thesedisorders may be to stimulate the local production of indigenousextracellular adenosine and NFs.

After acute injuries the extracellular concentration of the purineguanosine is higher than that of adenosine and, unlike adenosine,remains elevated for extended periods. Extracellular guanosine hassignificant neurotrophic and neuroprotective effects, includingstimulating the release of adenosine from cells and increasing synthesisof NFs. So guanosine may ameliorate the effects of acute CNS injury. Thesynthetic purine derivative AIT-082 mimics many of the biologicaleffects of guanosine. Therefore, it was investigated whether AIT-082might also exhibit the same activities and hence exert neuroprotectiveeffects. Specifically, it was determined whether: (1) AIT-082 protectsagainst long term excitotoxic neuronal damage induced by NMDA, in vitroand in vivo; and (2) the neuroprotective properties of AIT-082 aremediated through its effect on astrocytes.

The exposure of cultured astrocytes to AIT-082 for one hour caused adose-dependent increase of the outflow of radioactive adenine-basedpurines, which returned to the basal value two or three hours after theend of the drug treatment. The experiments were carried out on culturedastrocytes of rat hippocampus at the fourth day in vitro (DIV), obtainedfrom primary cultures replated in 35 mm Petri dishes (2×10 cells/dish)and preloaded with [³H]-adenosine (5×10⁻⁸ M for a specific activity of20.0 Ci/mM) for 30 minutes at 37° C.

The results are shown in FIG. 28. This shows the dose-dependent increaseof the outflow of radioactive adenine-based purines for a limited periodafter exposure.

The effect of GTP and guanosine on proportional release of radioactivelylabeled adenine nucleosides and nucleotides from rat cultured astrocytesis shown in Table P.

The exposure of astrocytes to GTP (300 μM) for one or three hourssignificantly stimulated the release of all adenine nucleotides, inparticular that of [³H]ADP and [³H]AMP. The release of [³H]adenosine andthat of [³H]hypoxanthine was markedly reduced. Conversely, guanosine(300 μM) did not affect the release of radioactively labeled adeninenucleotides but it increased the outflow of [³H]adenosine and to agreater extent that of [³H]inosine.

The effect of AIT-082 on the proportional release of radioactivelylabeled adenine nucleosides and nucleotides from rat cultured astrocytesis shown in FIGS. 29A and 29B. Culture medium collected during the firsthour of the release was analyzed by high performance liquidchromatography (HPLC) and the radioactivity associated with thedifferent adenine compounds was measured and expressed as a percentageof the total radioactivity released. The results are shown for ATP, ADP,and AMP in FIG. 29A, and for adenosine, inosine, and hypoxanthine inFIG. 29B. The exposure of the cultures to AIT-082 increased the releaseof adenosine and inosine in a dose-dependent fashion, without effectingthe release of hypoxanthine and the adenine nucleotides ATP, ADP, orAMP. These results are very similar to those obtained by treating thecultures with guanosine (Table O).

TABLE O NMDA-Induced Toxicity in Cultured Rat Hippocampal Neurons 1 hour3 hours GTP, Guanosine, GTP, Guanosine, Control 300 μM 300 μM Control300 μM 300 μM ATP 0.37 ± 0.03 1.28 ± 0.13 0.71 ± 0.09 0.29 ± 0.04 0.54 ±0.05 0.39 ± 0.04 (0.78 ± 0.6)  (10.9 ± 1.1)* (5.5 ± 0.7) (4.7 ± 0.7)(4.4 ± 0.4)  (3.1 ± 0.32) ADP 0.43 ± 0.03 2.10 ± 0.31 0.84 ± 0.09 0.32 ±0.04 1.05 ± 0.09 0.50 ± 0.07 (9.0 ± 0.6)  (17.8 ± 2.6)** (6.5 ± 0.7)(5.3 ± 0.7)  (8.6 ± 0.7)** (4.0 ± 0.6) AMP 0.58 ± 0.05 2.76 ± 0.28 1.58± 0.22 0.53 ± 0.06 4.32 ± 0.48 1.37 ± 0.14 (12.2 ± 1.0)   (23.5 ± 2.4)**(12.3 ± 1.7)  (8.7 ± 1.0)  (40.0 ± 3.9)*** (11.9 ± 1.0)  Adenosine 0.43± 0.04 0.47 ± 0.06 1.53 ± 0.10 0.33 ± 0.04 0.31 ± 0.02 1.16 ± 0.15 (9.1± 0.8)  (4.0 ± 0.5)** (11.9 ± 0.8)* (5.4 ± 0.7)  (2.5 ± 0.2)**  (9.3 ±0.9)** Inosine 0.86 ± 0.10 1.65 ± 0.2  4.64 ± 0.51 0.69 ± 0.08 1.19 ±0.19 4.09 ± 0.41 (17.9 ± 2.1)  (14.0 ± 1.7)   (36.0 ± 3.9)** (11.4 ±1.3)  (9.7 ± 1.2)  (3.25 ± 3.3)*** Hypoxanthine 1.62 ± 0.15 2.20 ± 0.313.41 ± 0.20 3.41 ± 0.42 3.11 ± 0.45 3.29 ± 0.34 (33.9 ± 3.1)   (18.4 ±2.6)** (24.7 ± 1.5)* (56.4 ± 6.9)   (25.0 ± 3.7)**  (26.3 ± 2.7)**Radioactivity released in the culture medium and identified by HPLCanalysis is reported as cpm × 10³. In the parentheses the values areexpressed as proportional release, calculated as the percentage of thetotal [³H]-purines released. The values are the mean ± SEM of 4experiments * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (unpairedStudent's t test)

The dose response for AIT-082 and guanosine on nerve growth factor andS100β protein release from rat culture astrocytes is shown in FIG. 30.The release of nerve growth factor is shown in FIG. 30A, while therelease of S100β protein is shown in FIG. 30B. NGF was assayed by adouble-site ELISA, using a monoclonal anti-NGF antibody, coupled or notto β-galactosidase according to the method of I. D. Laviada et al.,“Phosphatidylcholine-Phospholipase C Mediates the Induction of NerveGrowth Factor in Cultured Glial Cells,” FEBS Lett. 364: 301-304 (1995).

The S100β protein was measured by using an ELISA as described in A.Aurell et al., “The S-100 Protein in Cerebrospinal Fluid: A Simple ELISAMethod,” J.Neurol. Sci. 89: 157-164 (1989).

Previous evidence indicated that AIT-082 or guanosine increased theexpression of mRNA for some neurotrophic factors in neurons and inastrocytes. The exposure of astrocytes to these periods for 24 hoursalso caused a dose-dependent increase of the release of NGF and S100βprotein, a calcium binding protein able to promote at nanomolarconcentrations neuronal differentiation and astrocyte proliferation. Thespontaneous rate of NGF secretion was about 6 pg/hr/2×10⁵ cells whereasthe release of S100β protein was about 40 fold higher.

The time course of AIT-082 induced release of NGF and S100β protein fromrat cultured astrocytes is shown in FIG. 31. The NGF and S100β proteinwere measured by removing the cultured medium for the ELISA assays ateach indicated time point. The release of S100β protein from astrocytespeaked within three hours of exposure to AIT-082 (100 μM), and thenprogressively decreased. In contrast, the release of NGF was onlyenhanced 12 hours after the exposure to the drug and reached a maximumat 24 hours.

FIG. 32 shows the effects of cycloheximide on AIT-082-evoked release onNGF and S100β protein from rat cultured astrocytes. To determine whetherthe release of the neurotrophic factors was linked to new proteinsynthesis, cultured astrocytes were exposed to an inhibitor of proteinsynthesis, cycloheximide (0.5 μg/ml). This treatment abolished thestimulatory effect of AIT-082 (100μ) on the release of NGF, whereas theaccumulation of S100β protein in the medium was only slightly modified.

To determine whether or not AIT-082 afforded in vitro neuroprotection,the effect of AIT-082 on toxicity induced by N-methyl-D-aspartate (NMDA)in cultured rat hippocampal neurons was studied. In primary cultures ofhippocampal neurons, a pulse of 10 min. with NMDA (100 μM) inducedwithin 24 hours a dramatic increase of dead cells, determined by Trypanblue staining or LDH release. Such an increase is consistent with thedeath of nearly 75% of the cultured neurons as shown in Table P.

TABLE P NMDA-Induced Toxicity in Cultured Rat Hippocampal Neurons Countof Dead Cells LDH (Trypan Blue Staining) (mOD/min) Basal 33.4 ± 5.5 22.5± 4.6 NMDA (100 μM) 401.4 ± 27.3 262.8 ± 13.4

To determine whether AIT-082 protected against neuronal toxicity inducedby NMDA, and whether astrocytes might be involved, cultured astrocyteswere exposed to AIT-082 for 6 hr. The drug was then removed and cultureswere kept in fresh medium for the following 20 hr. This conditionedmedium was then added to primary cultures of rat hippocampal neurons onthe seventh day in vitro, immediately after they had been exposed to atoxic pulse of NMDA.

The results are shown in FIG. 33. Conditioned medium removed fromastrocytes, which had been treated with AIT-082 (50-100 μM), reduced by50% the number of the dead neurons and the production of LDH caused byNMDA treatment.

To confirm this, the effect of anti-NGF antibody on AIT-inducedprotection of glial conditioned medium in cultured hippocampal neuronsdamaged by NMDA was determined. The results are shown in FIG. 34. Theneuroprotective activity of the conditioned medium from astrocytes wasstrongly reduced by adding a neutralizing antibody specific for NGF.

The effect of AIT-082 on in vivo neuroprotection was also assessed byassaying for glutamic acid decarboxylase (GAD) activity.

GAD activity in rat caudate nuclei was damaged in vivo by NMDA. Theresults are shown in FIG. 35. Increasing amounts of NMDA (25-300 nmoles)were unilaterally infused into caudate nuclei of rats. Eight to 10 daysafter the injections, glutarnic acid decarboxylase (GAD) activity wasassayed, as an index of the loss of GABA-ergic neurons. NMDA produced adose-dependent lesion, which at its maximal extension caused a reductionof GAD activity by 50% in comparison with that of contralateralsham-operated nuclei (control). FIG. 36 shows the serial frontalsections across the extension of the caudate nuclei from a rat locallyinfused with 200 nmoles of NMDA. The necrotic areas are in white,whereas the shadow is the surrounding edema GAD activity was evaluatedby measuring the ex novo formation of [³H]GABA from dissected striataexposed to 1 μCi of [³H]glutamate for 1 hour at 37° C. GABA was assayedby HPLC with fluorescence detection and the [3H]label associated withGABA was measured.

The effect of local administration of AIT-082 on NMDA-induced unilaterallesion of rat striatum is shown in FIG. 37. The local adninistration of300 mnoles of AIT-082, coinfused with 200 nmoles of NMDA, almostcompletely prevented the loss of GAD activity.

The effect of systemic administration of AIT-082 on GAD activity in ratcaudate nuclei damaged by NMDA is shown in FIG. 38. AIT-082 wasadministered to rats by daily intraperitoneal injection for seven days.This treatment nearly completely preserved GAD activity in the striatadamaged by a local injection of 200 mnoles of NMDA.

These results were confirmed by magnetic resonance imaging (MRI). TheMRI results are shown in FIG. 39; Table Q shows the identification ofeach sample for which MRI results are shown in FIG. 39. T-2 weighted MRIphotos of transverse brain 3-mm thick slices were performed(TR/TE=7400/115 ms; matrix 483×1024; FLV 250). Magnetic resonance imageswere acquired using 1.5 T scanner (Vision, Siemens-Erlangen). Thecontrol injection of saline did not produce any change (A, B). A focalhyperintensity at the NMDA injection site was observed in brain slicesafter two days (C) and decrease in the hyperintensity was observed afternine days (D). The brightness of the lesion disappeared when AIT waslocally co-injected with NMDA (E, F), and it was significantly reducedwith systemic (intraperitoneal) administration of AIT-082 (60 mg/kg) (G,H). All animals survived the surgery and continued to receive treatmentfor an additional three days, at which time they were sacrificed andtissues were collected as described above.

TABLE Q MRI of Rat Striatum (FIG. 39) Days After Injection in the LeftStriata 2 Days 9 Days Saline A B NMDA (200 mmoles) C D AIT-082 + NMDA EF (Local co-injected) AIT-082 (Systemic i.p.) G H

In conclusion, in cultured astrocytes AIT-082, like guanosine, induce alarge increase of the extracellular levels of adenine nucleosides. Thiscontributes to the astrocyte-mediated neuroprotective effect of AIT-082.

In addition to a direct activity of neurons, AIT-082 exerts a potentneuroprotective activity by stimulating astrocytes to synthesize andrelease neurotrophic factors, such as NGF. S100β protein can play a rolein the AIT-082-mediated neuroprotection. It may be involved in a cascadeof positive feedback mechanisms which raise NGF production.

EXAMPLE 37 Effect of N-4-CARBOXYPHENYL-3-(6OXOHYDROPURIN-9-YL)Propanamide on Growth Factor mRNA Levels Following Splnal CordHemisection

To determine the effect of the administration ofN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide on neurotrophicfactor levels in the spinal cords of rats, the levels of suchneurotrophic factors were measured under various conditions.

Methods

T8 Lesion Surgery

Male Wistar rats (250 g) anesthetized with ketamine/xylazine receivedeither a partial laminectomy (sham operated control) or full laminectomyand unilateral transection of the spinal cord with a miniscalpel at thelevel of the eighth thoracic vertebrae. The animals were assigned intofour groups: two control groups, one group receivingN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide (20 mg/kg day) inthe drinking water and the other receiving no treatment. The two othergroups were both lesion groups of untreated and treated withN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide. A lesion wasjudged successful by the complete loss of withdrawal reflex 24 hourspost surgery. At the end of the treatment period the animals wereeuthanized and perfused with saline. A 2 cm segment of cord was takenaround the laminectomy site, called the lesion sample. Samples were alsotaken 2 cm above and 2 cm below the lesion (rostral and caudal sectionsrespectively). A diagram of the lesion regions is shown in FIG. 40.

RNA Extraction and RT-PCR

Total RNA was extracted from coded unfixed spinal tissue samples usingTRIzol reagent (Gibco-BRL). Total RNA (3 μg) was reversed transcribedusing a recombinant MMLV reverse transcriptase (RT) called SuperscriptII (Gibco-BRL) in a 20 μl reaction primed with oligo-dT₁₈ (MOBIX) usingbuffer and dithiothreitol supplied with the enzyme. Two aliquots (1 and2.5 μl) of the RT mixture were amplified in a mixture containing 0.2 mMdNTPs, 1×PCR buffer, 1.5 mM MgCl₂, Taq polymerase (Gibco-BRL) and 0.1 μgof sense and antisense primer (MOBIX). The quantitation of products wasdone from ethidium bromide stained gels using an LKB laser scanner usingthe ratio of the 1 μl to 2.5 μl replicates to ensure that thresholdfluorescence had not been reached. All samples were read in theexponential phase of the amplification curve for the primer set. Allthree primers sets were run from the same RT sample. The equivalence ofthe amount of RNA in each of the samples was corrected for theexpression of the housekeeping gene, G3PDH.

Results

RT-PCR was used to measure the mRNA levels of 3 neurotrophic factors,CNTF, BDNF, and NT-3 in 3 sections of the spinal cord as illustrated inFIGS. 41-43, after 3 or 7 days of treatment withN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide. The levels ofgene expression were normalized to G3PDH expression. The results areexpressed as relative ng of DNA after RT-PCR which are considered toreflect the levels of mRNA expression in the original RNA samples.

Rostral to the lesion, 3 days of treatment withN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide significantlyincreased the levels of BDNF and CNTF mRNA in sham-operated animals overthe lesion animals. This trend was reversed for BDNF after 7 days oftreatment with the compound when the relative levels of mRNA expressionwere significantly higher in the lesioned animals treated with thecompound than in the lesioned animals that had not been treated with thecompound. The compound, after 7 days, increased CNTF mRNA levelsrelative to lesion alone (p=0.056). However, in the control animals, thedrug treatment induced a robust increase (p=0.017) in CNTF levels overwater alone. At the rostral level, the surgical treatment alone did notsignificantly alter CNTF mRNA levels. At the level of the lesion,treatment with the compound increased mean CNTF mRNA levels over controlanimals, although not statistically so. There were no significantdifferences due to surgery or drug treatment at this level.

Caudal to the lesion for the control animals, NT-3 mRNA was decreasedafter 3 days of treatment with the compound. BDNF mRNA was higher in thesham-operated animals than in the lesioned animals when both groupsreceived the compound. There were no significant effects with 7 days oftreatment with the compound post surgery.

The levels of mRNA at the lesion site for the neurotrophins are shown inFIG. 41 (FIG. 41a, after 3 days of treatment; FIG. 41b, after 7 days oftreatment). The animals were treated for 3 (FIG. 41a) or 7 days (FIG.41b) as indicated. The error bars represent S.E.M.s (n=3 or 4). Therewere no significant differences between treatment groups. The mRNAlevels shown in FIG. 41 were measured by RT-PCR on a 2 cm portion of thespinal cord at the T8 lesion site as described above.

The effects of the compound on levels of mRNA rostral to the lesion areshown in FIG. 42 (FIG. 42a, after 3 days of treatment; FIG. 42b, after 7days of treatment). The animals were treated for 3 (FIG. 42a) or 7 days(FIG. 42b) as indicated. The mRNA levels were measured by RT-PCR on a 2cm portion of the spinal cord 1 cm up from the T8 lesion site asdescribed above. Statistical significance was determined by one-wayAnova testing. Error bars represent S.E.M.s (n=3 or 4). The level ofmRNA for CNTF in the sham-lesioned animals treated with the compoundwere significantly different from sham-lesioned animals treated onlywith water (p<0.05) after 7 days of treatment. The level of mRNA forBDNF was also significantly different when lesioned animals treated withthe compound were compared with lesioned animals treated only with water(p<0.05) after 7 days of treatment. The level of BDNF mRNA after 3 daysof treatment in control animals (i.e. sham-lesioned animals) treatedwith the compound was significantly different from lesioned animalstreated with the compound (p<0.05). Finally, the level of CNTF mRNA at 3days of treatment in control animals treated with the compound was againsignificantly different from lesioned animals treated with the compound(p<0.01).

The effects of the compound on levels of mRNA caudal to the lesion areshown in FIG. 43 (FIG. 43a, after 3 days of treatment; FIG. 43b, after 7days of treatment). The mRNA levels were measured by RT-PCR on a 2 cmportion of the spinal cord I cm down from the T8 lesion site asindicated above. The animals were treated for 3 (FIG. 43a) or 7 days(FIG. 43b) as indicated. Statistical significance was determined byone-way Anova testing. Error bars represent S.E.M.s (n=3 or 4). Thelevel of NT-3 mRNA was lower after treatment with the compound insham-lesioned animals as compared with sham-lesioned animals not treatedwith the compound (p<0.05) after 3 days of treatment.

In conclusion, the compound appeared to suppress neuronal production ofNT-3 at three days of treatment in the cords of control (sharn-lesioned)animals. While the effect was significant only in the segment caudal tothe lesion, the trend was seen in all three segments of the cord. Sevendays of treatment with the compound resulted in increased CNTF in thecords of control animals, though statistical significance was seen onlyin one segment. BDNF was markedly elevated in lesioned animals treatedwith the compound in the cord rostral to the lesion. The elevatedexpression seen in this segment may reflect the increased population ofneuronal cells that were present at this level of the cord.

EXAMPLE 38 AIT-082 Effects on Neurotrophic Factor Production in theBrain

Studies in this example examine whether AIT-082: (1) promotes a survivalof basal forebrain: cholinergic neurons after fimbria-fornix lesions;(2) influences the production of neurotrophic factors in the brain; and(3) influences cellular and neurotrophin changes in the aged brain. Itwas found that AIT-082 influences levels of the nervous system growthfactors BDNF, GDNF, and NT-3 in specific regions of the brain underspecific conditions.

Promotion of Survival of Basal Forebrain Cholinergic Neurons AfterFimbria-Fomix Lesions by AIT-082

This model is the gold standard for examining the effects ofneurotrophic factors, neurotrophic factor-releasing agents, andneurotrophic factor analogues in the central nervous system (CNS).Fimbria-fornix transections (FFT) are performed to induce thedegeneration of basal forebrain cholinergic (BFC) neurons. In theabsence of treatment, the number of BPC neurons is reduced to ˜25% ofnormal numbers two weeks after the lesion, as identified by cholineacetyltransferase (ChAT) immunolabeling or p75 (low affinityneurotrophin receptor) immunolabeling. The infusion of nerve growthfactor completely prevents the degeneration of these neurons. In thisexperiment, animals received AIT-082 to determine whether it wouldprevent lesion-induced degeneration of BFC neurons.

Methods

Adult female F344 rats received an intraperitoneal (IP) injection ofAIT-082 (30 mg/kg) or saline one hour prior to receiving a unilateralfimbria-fornix transection (FFT) (n=6 per group). All animals survivedthe surgery and continued to receive IP injections of AIT-082 or vehicleevery three days following the lesion. All animals were perfused withfixative at 14 days post-lesion and brains were sectioned and processedfor choline acetyltransferase (ChAT) and p75 immunoreactivity. Foranalysis, three sections, 240 μm apart, were taken at the level of themedial septum and ChAT positive cells were counted both ipsilateral andcontralateral to the lesion.

Results

Vehicle-infused control animals demonstrated a characteristic 70-75%loss of ChAT-positive cells on the side ipsilateral to the lesion.Administration of AIT-082 did not reduce lesion-induced loss ofChAT-labeled cells in the BFC region (FIG. 44). For the results of FIG.44, ChAT-positive cells were counted in three sections, 240 μm apart.Data was expressed as a ratio of cell counts ipsilateral to the lesiondivided by counts contralateral to the lesion. Animals were givenintraperitoneal injections of either AIT-082 (30 mg/kg/day; n=6) orsaline (n=6) every three days beginning on the day of the lesion.Injections were administered throughout the post-lesion survivalinterval of 14 days. No significant differences were seen in the numberof ChAT positive cells on the lesion side as a result of the AIT-082treatment. NGF data was obtained from Barnett et al. (Exp. Neurol.110:11-24(1990)) using mouse NGF (25 μg/ml) and illustrates a robustrescue effect following the FFT lesion.

Influence of Neurotrophic Factors in the Brain by AIT-082

Previously it had been shown that AIT-082 influences expression of thegrowth factors NGF, neurotrophin-3 (NT-3) and fibroblast growth factor 2(FGF-2 or bFGF) but not brain-derived neurotrophic factor (BDNF) invitro and in vivo. Previous in vivo studies used RT-PCR, which issemi-quantitative and is not clearly related to levels of neurotrophinproteins in vivo. Therefore, it was determined whether AIT-082upregulates NGF, BDNF, NT-3, and GDNF protein expression (by ELISA) inthe brain and spinal cord.

Influence of AIT-082 on the Production of Neurotrophic Factors in theIntact Adult Brain.

Methods

Adult female F344 rats were given either AIT-082 (doses: 30 mg/kg/day ora higher dose of 30 mg/ml (˜3,000 mg/kg/day); n=6 rats per group) intheir drinking water, or water alone, as a vehicle for control. Theduration of the study was seven days, at which time the rats wereanesthesized and perfused with ice-cold saline. The brains of the ratswere rapidly removed. The brains were placed on a chilled glass plateand dissected into the following regions: basal forebrain, frontalcortex, parietal cortex, cerebellum, spinal cord, and hippocampalformation. Tissues were stored at −70° C. until assayed in a specifictwo-site ELISA for either NGF, BDNF, NT-3, or GNDF.

The results are shown in FIGS. 45 (BDNF), 46 (NT-3), 47 (GDNF) and 48(NGF). For the results shown in FIGS. 45-48, animals were given onlywater (VEH, n=6) or water with AIT-082 at either 30 mg/kg/day (n=6) or30 mg/ml (n=6) for seven days. The levels of each growth factor weredetermined in the frontal cortex (F. Cor.), parietal cortex (P.Cor.),hippocampal formation (Hipp.), basal forebrain (B.F.), cerebellum(Cer.), or spinal cord (Cord) using a specific two-site ELISA for eachgrowth factor. An asterisk (*) indicates that the results weresignificantly different (p<0.05) from vehicle-treated animals; a # signindicates that the results were significantly different from animalstreated with AIT-082 at 30 mg/ml (p<0.05).

All animals receiving the compound AIT-082, at high and low doses,appeared normal and showed no overt signs of weight loss during the oneweek of administration. AIT-082 at 30 mg/kg/day increased BDNF levels infrontal cortex more than twofold over vehicle controls (FIG. 45). Thiseffect was not seen in animals treated with AIT-082 at 3000 mg/kg/day.

AIT-082 treatment also increased BDNF levels twofold in the spinal cordover vehicle controls, although this effect did not achieve significance(p>0.05). AIT-082 treatment of 30 mg/kg/day increased NT-3 levels incerebellum over vehicle controls (FIG. 46). This effect was again absentin animals receiving 3000 mg/kg/day. AIT treatment (at 3000 mg/kg/daydose) increased GDNF levels in the basal forebrain and spinal cord abovevehicle treated control levels (FIG. 47). No changes were seen in NGFlevels as a result of the treatment (FIG. 48).

In conclusion, AIT-082 significantly elevated levels of three trophicfactors in specific regions of the CNS when administered orally. BDNFlevels increased in frontal cortex, a region implicated in Alzheimer'sdisease, and NT-3 levels increased in the cerebellum, a regionimplicated in several other neurological disorders of balance andcoordination.

Modulation of Neurotrophic Factor Levels by AIT-082 in the LesionedAdult Brain

Most neurological disorders in which AIT-082 therapy would beadministered represent conditions of neuronal injury. Expression of somegrowth factors is selectively increased after brain injury, suggestingthat AIT-082 may be more effective at modulating growth factor activityunder injured conditions. To explore this question, the experiment abovewas repeated in animals with lesions of BFC neurons.

Methods

Adult female rats were given AIT-082 (30 mg/kg/day) in their drinkingwater or drinking water alone (n=6 for each group) for seven days priorto receiving a bilateral fimbria fornix transection (FFT).

Results

The results are shown in FIGS. 49 (BDNF), 50 (NT-3), 51 (GDNF) and 52(NGF). For the results shown in the Figures, animals were given onlywater (VEH, n=3) or water with AIT-082 at 30 mg/kg/day (n=3). Levels ofthe growth factor were determined in frontal cortex (F.Cor.), parietalcortex (P.Cor.), hippocampal formation (Hipp.), basal forebrain (B.F.),cerebellum (Cer.), or spinal cord (Cord) using a specific two-site ELISAfor each growth factor. An asterisk (*) indicates a result significantlydifferent from vehicle treated animals (p<0.05).

AIT-082 treatment again increased levels of BDNF in frontal cortexalmost threefold over vehicle controls (FIG. 49). However, statisticalsignificance was not achieved at a high variability in BDNF levels. Inaddition, AIT-082 treated FFT-lesioned animals exhibited threefoldincreased in NT-3 levels within the basal forebrain compared to vehiclecontrols (FIG. 50). In the fimbria-fornix lesioned animals, AIT-082treatment increased GDNF levels within the basal forebrain above vehiclecontrols (FIG. 51). No significant increases in NGF levels were found asresult of the treatment (FIG. 52).

In conclusion, AIT-082 significantly elevates levels of two neurotrophicfactors in specific regions of the injured CNS when administered orally.BDNF levels increase in frontal cortex, consistent with the resultsgiven above. This region is implicated in Alzheimer's disease. Inaddition, NT-3 and GDNF levels increase in the cholinergic basalforebrain, a region of substantial cell loss in Alzheimer's disease.

Influence of AIT on Cellular and Neurotrophin Changes in the Aged Brain

Previously, it has been shown that AIT-082 improves memory in aged ratsand increases neurotrophin mRNA levels. Aging may represent a period ofparticular neuronal vulnerability to degeneration, including the changesof Alzheimer's disease. Thus, aged animals merit special independentstudy. This experiment examined AIT-082 induced alterations inneurotrophin levels in aged animals.

Methods

Aged female Fischer 344 rats (obtained from National Institute on Aging;24 months on arrival) were given AIT-082 (30 mg/kg/day) in theirdrinking water or drinking water alone, for a period of 30 days (n=5rats per group). After 30 days, animals were sacrificed and freshtissues were dissected as described above. An additional set of younganimals (n=5) was processed along with the aged rats for comparison.

Results are shown in FIGS. 53 (BDNF), 54 (NT3), 55 (GDNF), and 56 (NGF).As described above, for the results shown in these figures, aged rats(24 months of age) were given water (vehicle control; n=5) or water withAIT-082 (at 30 mg/kg/day; n=5 for 30 days). A separate group of younguntreated animals was also included (n=5). Levels of each growth factorwere determined in frontal cortex (F.Cor.), parietal cortex (P. Cor.),hippocampal formation (Hipp.), basal forebrain (B.F.), cerebellum (Cer.)or spinal cord (Cord) using a specific two-site ELISA. An asterisk (*)indicates that the results are significantly different from those withyoung animals (p>0.05); a # sign indicates that the results aresignificantly different from those with untreated aged animals (p>0.05).

Within the hippocampus, there was a significant age-related decline inNT-3 levels of aged control animals compared to young animals.Significantly, AIT-082 treatment restored NT-3 protein levels to thoseof young animals (FIG. 54). Slight increases in GDNF levels were notedin the parietal and frontal cortex as a result of aging; however,AIT-082 treatment restored GDNF protein levels to those of young animals(FIG. 55). AIT-082 treatment in the aged animal had no effect on BDNF(FIG. 53) or NGF (FIG. 56) in any of the brain regions examined. Inconclusion, AIT-082 reversed age-related declines in hippocampus NT-3levels. The hippocampus is predominantly involved in Alzheimer'sdisease.

Generally, in conclusion, this data indicates that AIT-082 can increaseneurotrophin levels in specific brain regions. Although AIT-082 failedto show a neuroprotective effect in the fimbria-fornix model, neuronaldegeneration in the fimbria-fornix model is most sensitive to NGF. Ofthe growth factors assayed, AIT-082 influenced BDNF, NT-3, and GDNFlevels but not those of NGF, thereby potentially accounting for the lackof neuroprotection in the fimbria-fornix model.

A compound that can be administered orally to modulate neurotrophicfactors in the CNS is an important finding and generally supportsseveral previous in vitro and in vivo studies performed with AIT-082.Findings of the study of this Example suggest a mechanistic basis forthe neuroprotective actions of AIT-082, by demonstrating specificincreases in levels of BDNF, NT-3, and GDNF as the result of treatment.In particular, AIT-082 increases BDNF protein levels in the frontalcortex, a brain region thought to play an important role in severalcognitive tasks. Further, BDNF has been shown in previous studies tomodulate synaptic plasticity, which could enhance cognition.

The finding that AIT-082 augments NT-3 in the hippocampus supports itspotential utility in Alzheimer's disease. Further, the finding that NT-3levels are also increased in the cerebellum broadens the potentialtherapeutic targets of AIT-082.

Levels of the potent motor neuron and dopaminergic neuron growth factorGDNF were also significantly enhanced in this study in the brain stemand spinal cord. These findings raise a possibility that AIT-082 may beof therapeutic benefit in Parkinson's disease (the second most commonneurodegenerative disorder), amyotrophic lateral sclerosis (Lou Gehrig'sdisease), and spinal cord injury.

EXAMPLE 39 Production of Neurotrophic Factors Stimulated by AIT-082 inRat Cultured Astrocytes

Guanine-based purines, released in large amounts from astrocytes(Ciccarelli et al., Glia 25: 93-98 (1999)) have been recently recognizedas extracellular signaling molecules, able to exert important trophiceffects on both neurons and glia (Rathbone et al., Drug Dev. Res. 45:356-372 (1998)). The hypoxanthine derivative AIT-082, also known asN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl)propanamide or4-(3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl)amino) benzoic acid,shares some trophic effects with guanine-based purines in vitro.Moreover, AIT-082, administered in vivo, restores age- andexperimentally-induced working memory deficits in rodents (Glasky etal., Pharm. Biochem. Behav. 47: 325-329 (1994)). In these activities,the production of neurotrophic factors (NFs) could be involved(Middlemiss et al., Neurosci. Lett. 199: 1-4 (1995)).

Today, NFs are considered as potential drugs for the experimentaltherapy of neurodegenerative disorders of the brain, owing to theircapability of enhancing neuronal recovery and survival. However, theclinical potential of NFs is undermined by their inability to cross theblood-brain barrier and by their induction of severe side effects aftersystemic administration. Thus, one possible therapeutic approach is toinduce a drug-mediated increase in the local production of endogenousNFs in the brain, assuming astrocytes as target cells that produce largeamounts of either neurotrophins (nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), orneurotrophin 4/5 (NT-4/5)) or pleiotrophins (transforming growth factorβ (TGFβ), fibroblast growth factors (FGFs), or S100β protein).

In the studies reported in this Example, it was investigated whether:(1) AIT-082, like guanosine, stimulated the production of some NFs suchas NGF or TGFβ from rat cultured astrocytes; and (2) whether the NFsreleased from cultured astrocytes following AIT-082 stimulation wereprotective against excitotoxic damages caused by NMDA in culturedneurons.

The dose response of NGF and TGFβ₂ from rat cultured astrocytes asinduced by guanosine is shown in FIG. 57 The exposure of astrocytes toguanosine for 24 hours caused a dose-dependent increase of the releaseof NGF and TGFβ₂. NGF and TGFβ₂ levels were assayed in the culturemedium of the astrocytes by specific ELISA assays using kits from Roche(Germany) and Promega (USA) respectively. NGF basal levels were 56.3±3.2pg/ml. TGFβ₂ levels were 9.1±0.06 pg/ml.

Western blot analysis of cytosolic proteins from cultured astrocytes wasperformed to detect the factors NGF and TGFβ₂. The results are shown inFIG. 58A for NGF at 6 and 12 hours and in FIG. 58B for TGFβ₂ at 6 and 12hours. These results indicate that guanosine increased the intracellularcontent of NGF and TGFβ₂ in time-dependent fashion.

The results shown in FIGS. 59A and 59B show the involvement of themitogen-activated protein kinase cascade and ex novo protein synthesisin the guanosine-induced effect. In FIG. 59A, the effect of twoinhibitors of the mitogen-activated protein kinase cascade, wortmanninand PD098,059, is shown. In the results shown in this figure, 100 nMwortmannin or 10 μM PD098,059 were added to the cultures 30 minutesbefore guanosine and maintained for 24 hours together with 300 μMguanosine. In FIG. 59B, the effect of the protein synthesis inhibitorcycloheximide (CHX) is shown. In the results shown in this figure, theCHX was added to the cultures 30 minutes before guanosine and maintainedfor 24 hours together with 300 μM guanosine. The effects of MAP kinaseinhibitors or protein synthesis inhibitor are expressed as % of theeffect caused by guanosine on NGF and TGFβ₂ release assumed as equal to100%.

In FIG. 60, the activation of MAP kinases ERK1 (44 kDa) and ERK2 (42kDa) by guanosine is shown. Cultured astrocytes were harvested at 4° C.in lysis buffer specific for MAP kinase: 25 mM Tris buffer, pH 7.4,containing 150 mM NaCl, 100 μM sodium orthovanadate, 1.5 mM MgCl₂, 1.0mM EDTA, 1% NP40, 10% glycerol, 1 mM PMSF, 5 μg/ml leupeptin, and 10μg/ml aprotinin and sonicated and centrifuged at 14,000 rpm for 5 min.Aliquots of the supernatants were processed for the assessment ofprotein concentration (M. M. Bradford, “A Rapid and Sensitive Method forthe Quantitation of Microgram Quantities of Protein Using the Principleof Protein-Dye Binding,” Anal. Biochem. 72: 248-254 (1976)).Electrophoresis was performed in 12% SDS-PAGE, using 20 μg of totalprotein per lane and separated proteins were then transferred onto aPVDF membrane (Bio-Rad Laboratories, Italy). Membranes were firstincubated with polyclonal primary antibody (rabbit phospho-ERK1/2antibody, New England Biolabs, Germany; final dilution 1:1000) for 1hour and then with donkey anti-rabbit HPR-conjugated secondary antibody(Amersham, Italy; final dilution 1:5000) for another 1 hour, both atroom temperature. Immunocomplexes were visualized using the enhancingchemiluminescence detection system (ECL) (Amersham, Italy).

In FIG. 61, the dose-response curves of NGF and TGFβ₂ release from ratcultured astrocytes as the result of exposure to AIT-082 are shown. NGFand TGFβ₂ levels were measured in the same manner as for the resultsshown in FIG. 57. The results indicated that AIT-082 caused atime-dependent increase of NGF production by about 2.5-fold and TGFβ₂production by 1.7-fold, as compared to a control.

Western blot analysis of cytosolic proteins deriving from culturedastrocytes treated for 6 or 12 hours with 100 μM AIT-082 was performed.AIT-082 is able to promote the production of neurotrophic factors fromrat cultured astrocytes as measured by Western blot analysis. Theresults are shown in FIG. 62A for NGF and in FIG. 62B for TGFβ₂.Electrophoresis was performed in 12% (for NBF) or 15% (for TGFβ₂)SDS-PAGE (20 μg of total protein per lane). The separated proteins weretransferred onto a PVDF membrane (Bio-Rad Laboratories, Italy).Membranes were incubated (1 hr) with polyclonal primary antibody (rabbitanti-NGF, Santa Cruz Biotechnology, CA; final dilution, 1:100; rabbitanti-TGFβ₂, Santa Cruz Biotechnology; final dilution 250 ng/ml) and thenwith donkey anti-rabbit HRP-conjugated secondary antibody (Amersham,Italy; final dilution 1:5000 (1 hr), both at room temperature.Immunocomplexes were visualized by the enhancing chemiluminescencedetection system (ECL). AIT-082 (100 μM) increased in time-dependentmanner the intracellular levels of both trophins.

Moreover, at the same dose, AIT-082 did not stimulate ex novo synthesisof S100β protein. This finding confirmed previous data obtained by ELISAassay on astrocyte culture medium, demonstrating that AIT-082 increasedonly early release of this pleiotrophin from astrocytes into the culturemedium.

At the same time, it was found that AIT-082, at its most active dosage(100 μM) induced, 5 min after the exposure of cultured astrocytes, themaximal activation of the molecular enzyme cascade. This activation wasno longer evident after 10 minutes or 20 minutes of stimulation. Inparticular, by a selective Western blot analysis, it was found thatAIT-082 stimulated the phosphorylation of MAP kinases ERK1 (44 kDa) andERK2 (42 kDa), corresponding to the activated isoforms of the enzymes,in astrocyte cytosolic proteins. This effect is likely independent ofthe activity of growth factors, such as NGF, that also act through thismolecular pathway. These results are shown in FIG. 63. Culturedastrocytes were harvested at 4° C. in lysis buffer specific for MAPkinase: 25 mM Tris buffer, pH 7.4, containing 150 mM NaCl, 100 μM sodiumorthovanadate, 1.5 mM MgCl₂, 1.0 mM EDTA, 1% NP40, 10% glycerol, 1 mMPMSF, 5 μg/ml leupeptin, and 10 μg/ml aprotirin and sonicated andcentrifuged at 14,000 prm for 5 min. Aliquots of the supernatants wereprocessed for the assessment of protein concentration (Bradford, 1976).Electrophoresis was performed in 12% SDS-PAGE, using 20 pg of totalprotein per lane and separated proteins were then transferred onto aPVDF membrane (Bio-Rad Laboratories, Italy). Membranes were firstincubated with polyclonal primary antibody (rabbit phospho-ERK1/2antibody, New England Biolabs, Germany; final dilution 1:1000) for 1hour and then with donkey anti-rabbit HPR-conjugated secondary antibody(Amersham, Italy; final dilution 1:5000) for another 1 hour, both atroom temperature. Immunocomplexes were visualized using the enhancingchemiluminescence detection system (ECL) (Amersham, Italy).

The AIT-082-induced accumulation of both trophins, mainly that of NGF,was partly inhibited by the pretreatment of the culture with wortmannin,an inhibitor of phosphatidyl inositol 3-kinase, an enzyme involved inMAP kinase cascade activation by G-protein coupled receptors, orPD098,059, an inhibitor of MAP kinase kinase activity as well as by aknown inhibitor of protein synthesis, cycloheximide (CHX). The resultswith these inhibitors are shown in FIG. 64. In these experiments, 100 nmwortmannin or 10 μM PD098,059 or 500 ng/ml CHX were added to thecultures 30 min before AIT-082 and maintained for 24 hours together with100 μM AIT-082. The effects of the MAP kinase inhibitors (left panel) orcycloheximide (right panel) are expressed as % of the effect caused byAIT-082 on NGF or TGFβ₂ release assumed as equal to 100%.

It was next evaluated whether the addition of conditioned medium (CM)deriving from astrocyte cultures treated with 100 μM AIT-082 generatedprotection against damage induced by a toxic pulse with 100 μM NMDA incultured neurons. Previously, it was demonstrated that the addition ofCM, obtained as indicated above, was protective against NMDA-inducedtoxicity in hippocampal neurons. This protective effect was ascribedmainly to NGF production caused by astrocyte treatment with AIT-082.Indeed, if the astrocyte-derived CM was added to hippocampal neuronstogether with specific NGF-neutralizing antibodies or TGFβ₂-neutralizingantibodies, a significant reduction of the protective effect wasobserved. In further experiments, the co-addition of CM and specificantibodies to S100β protein (100 ng/ml) to NMDA-damaged hippocampalneurons did not modify CM-induced neuroprotection.

In these experiments, cultured neurons were prepared from rat cerebralcortex. In these cultures the NMDA pulse (100 μM for 10 min) provokedabout 80% neuronal death (measured by Trypan blue cell staining or LDHactivity). Separately, cultured astrocytes were treated for 6 hours withAIT-082. Then, the medium containing the drug was replaced with freshmedium without the drug and the astrocyte cultures were maintained inthis medium for the next 24 hours. This medium represented the CM andwas added to neurons soon after the toxic pulse with NMDA. The additionof CM partly counteracted the toxic effects caused by NMDA, reducing theneuronal damage by about 50%. If the medium was boiled for 20 min at100° C. prior to addition to NMDA-damaged neurons, it completely lostthe protective activity. This finding suggested that the AIT-082-inducedprotection was likely mediated through a heat-sensitive factor, likely aprotein. To verify whether the neuroprotection caused by AIT-082 wasascribable to the production of TGFβ₂ or NGF, antibodies to TGFβ₂ or toNGF (100 ng/ml) were added together with CM. This treatment also reducedthe neuroprotective effect of the CM by about 40%.

The experimental scheme for the use of conditioned medium (CM) is shownin FIG. 65. The effects of NMDA are shown in FIG. 66A on cortical cellsand in FIG. 66B for hippocampal cells. In FIGS. 66A and 66B, the effectof 100 μM NMDA on the cell cultures is shown; NMDA increased the numberof dead cells as measured by Trypan Blue staining and increased therelease of LDH activity. In FIG. 67A, the protection provided by CMalone or together with 100 μM AIT-082 for hippocampal cells is shown,together with results when anti-NGF antibody is added together with CMand AIT-082. Similarly, in FIG. 67B, the protection provided by CM aloneor together with 100 μM AIT-082 for hippocampal cells is shown, togetherwith results when anti-TGFβ₂ antibody is added together with CM andAIT-082. These results indicate that AIT-082 provides protection againstthe excitotoxic effects of NMDA, and this protection is partiallycounteracted by antibodies against TGFβ₂ or NGF, suggesting that theprotection is mediated by these factors. Specifically, CM alone reducedcell damage by about 20%. The combination of AIT-082 and CM reduced celldamage by approximately 70-80%, and this reduction in cell damage wascounteracted to a significant degree when antibodies to NGF (FIG. 67A)or TGFβ₂ (FIG. 67B) were added.

Since it has been previously found that the stimulation of A₁ adenosinereceptor increased the production of trophic factors from astrocytes andthat AIT-082 is able to increase the extracellular adenosine levels inthe culture medium of astrocytes, it was investigated whether theneuroprotective effect of CM was modified by adding, at the same time,CM and a selective blocker of A₁ adenosine receptor,8-cyclopentyl-1,3-dipropylxanthine (DPCPX)(100 nm) to the injuredcortical neurons. DPCPX did not modify the neuroprotective effect ofastrocyte-derived CM. Conversely, when DPCPX (100 nm) was added toastrocyte cultures together with AIT-082, the neuroprotective effect ofthe resulting CM deriving from the culture of the astrocytes withAIT-082 was reduced by only 20%, as compared to the neuroprotectiveeffect of CM derived from astrocytes exposed to AIT-082 alone (assumedas equal to 100%).

To summarize the conclusions of this Example, AIT-082 stimulatesastrocytes to synthesize and release NFs. Indeed, this compound dose-and time-dependently increased the production of NGF and TGFβ₂ fromastrocytes. NGF accumulation in the culture medium was greater than thatof TGFβ₂ as compared with control. Moreover, the enhancement in therelease of both NFs needs some hours of cell exposure to the drug andthe activation of ex novo protein synthesis. The increased production ofNFs caused by AIT-082 involves the activation of MAP kinase cascade,whose rapid onset likely suggests a direct effect of this drug on thissignal transduction pathway. However, specific receptors for thishypoxanthine derivative are yet unknown. The AIT-082 induced productionof NGF and TGFβ₂ by astrocytes is directly involved in theneuroprotective effect exerted by this agent in vivo.

EXAMPLE 40 Summary of Effects of AIT-082 on Production of mRNA EncodingGrowth Factors and on Production of Growth Factor Proteins

The constellation of effects of AIT-082 on production of mRNA encodinggrowth factors and on production of growth factor proteins is shown inTable R. The results summarized in Table S show both transcriptional andtranslational effects on growth factors that are in the categories ofneurotrophins, pleiotrophins, members of the S100 family of EF handcalcium binding proteins, and members of the TGFβ superfamily. Asindicated, effects have been shown at both the mRNA (transcriptional)and protein (translational) levels.

TABLE R Constellation of Effects of AIT-082 on Production of mRNA Codingfor Growth Factors and Production of Growth Factor Proteins CulturedLesioned Normal Lesioned Cells Normal Brain Aged Brain Brain Spinal CordSpinal Cord Neurotrophins NGF ±/+ − ± ±/− − nt NT-3 ± + + ±/+ − ± BDNFnt + + ±/+ + ± Pleiotrophins bFGF ±/+ nt ± ± nt nt CNTF nt nt nt nt ± ±S100 Family (EF Hand Calcium-Binding Proteins) S100β + nt nt nt nt ntTGFβ Superfamily TGFβ₁ + nt nt nt nt nt GDNF nt + + + + nt ± = mRNA + =protein nt = not tested

Those skilled in the art will further appreciate that the presentinvention may be embodied in other specific forms without departing fromthe spirit or central attributes thereof. In that the foregoingdescription of the present invention discloses only exemplaryembodiments thereof, it is to be understood that other variations arecontemplated as being within the scope of the present invention.Accordingly, the present invention is not limited to the particularembodiments which have been described in detail herein. Rather,reference should be made to the appended claims as indicative of thescope and content of the present invention.

What is claimed is:
 1. A method for selectively and controllablyinducing the in vivo genetic expression of at least one naturallyoccurring genetically encoded molecule in a mammal comprising the stepof administering an effective amount of at least one carbon monoxidedependent guanylyl cyclase modulating purine derivative to the mammal.2. The method of claim 1 wherein the at least one naturally geneticallyencoded molecule stimulates neuritogenesis.
 3. The method of claim 2wherein the at least one naturally occurring genetically encodedmolecule that stimulates neuritogenesis is selected from the groupconsisting of neurotrophins, pleiotrophins, members of the S100 familyof EF hand calcium binding proteins, and members of the TGFβsuperfamily.
 4. The method of claim 3 wherein the at least one naturallyoccurring genetically encoded molecule that stimulates neuritogenesis isa neurotrophin.
 5. The method of claim 4 wherein the neurotrophin isselected from the group consisting of nerve growth factor (NGF), NT-3,and brain-derived neurotrophic factor (BDNF).
 6. The method of claim 5wherein the neurotrophin is NGF.
 7. The method of claim 5 wherein theneurotrophin is NT-3.
 8. The method of claim 5 wherein the neurotrophinis BDNF.
 9. The method of claim 3 wherein the at least one naturallyoccurring genetically encoded molecule that stimulates neuritogenesis isa pleiotrophin.
 10. The method of claim 9 wherein the pleiotrophin isselected from the group consisting of basic fibroblast growth factor(bFGF) and ciliary neurotrophic factor (CNTF).
 11. The method of claim10 wherein the pleiotrophin is bFGF.
 12. The method of claim 10 whereinthe pleiotrophin is CNTF.
 13. The method of claim 3 wherein the at leastone naturally occurring genetically encoded molecule that stimulatesneuritogenesis is a member of the S100 family of EF hand calcium bindingproteins.
 14. The method of claim 13 wherein the member of the S100family of EF hand calcium binding proteins is selected from the groupconsisting of S100β, p11, p9Ka, and calcyclin.
 15. The method of claim14 wherein the member of the S100 family of EF hand calcium bindingproteins is S100β.
 16. The method of claim 14 wherein the member of theS100 family of EF hand calcium binding proteins is p11.
 17. The methodof claim 14 wherein the member of the S100 family of EF hand calciumbinding proteins is p9Ka.
 18. The method of claim 14 wherein the memberof the S100 family of EF hand calcium binding proteins is calcyclin. 19.The method of claim 3 wherein the at least one naturally occurringgenetically encoded molecule that stimulates neuritogenesis is a memberof the TGFβ superfamily.
 20. The method of claim 19 wherein the memberof the TGFβ superfamily is selected from the group consisting of TGFβ₁and glial cell line-derived neurotrophic factor (GDNF).
 21. The methodof claim 20 wherein the member of the TGFβ superfamily is TGFβ₁.
 22. Themethod of claim 20 wherein the member of the TGFβ superfamily is GDNF.23. The method of claim 1 wherein the inducing of the in vivo geneticexpression of at least one naturally occurring genetically encodedmolecule occurs in astrocytes of the mammal.
 24. The method of claim 1wherein the inducing of the in vivo genetic expression of at least onenaturally occurring genetically encoded molecule activates themitogen-activated protein kinase cascade.
 25. The method of claim 1wherein the carbon monoxide dependent cyclase modulating purinederivative is selected from the group consisting of guanosine, inosinepranobex, and a compound of formula (I)

where n is an integer from 1 to 6 or of a salt or prodrug ester of acompound of formula (I) where n is an integer from 1 to
 6. 26. Themethod of claim 25 wherein the compound is a compound of formula (I)wherein n is an integer from 1 to
 6. 27. The method of claim 26 whereinn is 2 and wherein the compound isN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl)propanamide.
 28. The method ofclaim 1 wherein the effective amount of the at least one carbon monoxidedependent guanylyl cyclase modulating purine derivative produces atreating concentration of at least 1 μM.
 29. The method of claim 1wherein the at least one carbon monoxide dependent guanylyl cyclasemodulating purine derivative is orally administered to the mammal. 30.The method of claim 1 wherein the at least one carbon monoxide dependentguanylyl cyclase modulating purine derivative is administered to themammal by injection.
 31. The method of claim 1 wherein the mammal is ahuman.
 32. A method for the administration of at least one naturallyoccurring genetically encoded molecule to a mammal comprising the stepof selectably inducing the in vivo genetic expression of the molecule inthe mammal through the administration of an effective amount of at leastone carbon monoxide dependent guanylyl cyclase modulating purinederivative to the mammal to raise the concentration of the at least onenaturally occurring genetically encoded molecule in at least one tissueof the mammal and thus cause the administration of the at least onenaturally occurring genetically encoded molecule to the mammal.
 33. Themethod of claim 32 wherein the at least one naturally occurringgenetically encoded molecule stimulates neuritogenesis.
 34. The methodof claim 33 wherein the at least one naturally occurring geneticallyencoded molecule that stimulates neuritogenesis is selected from thegroup consisting of neurotrophins, pleiotrophins, members of the S100family of EF hand calcium binding proteins, and members of the TGFβsuperfamily.
 35. The method of claim 34 wherein the at least onenaturally occurring genetically encoded molecule that stimulatesneuritogenesis is a neurotrophin.
 36. The method of claim 35 wherein theneurotrophin is selected from the group consisting of NGF, NT-3, andBDNF.
 37. The method of claim 36 wherein the neurotrophin is NGF. 38.The method of claim 36 wherein the neurotrophin is NT-3.
 39. The methodof claim 36 wherein the neurotrophin is BDNF.
 40. The method of claim 34wherein the at least one naturally occurring genetically encodedmolecule that stimulates neuritogenesis is a pleiotrophin.
 41. Themethod of claim 40 wherein the pleiotrophin is selected from the groupconsisting of bFGF and CNTF.
 42. The method of claim 41 wherein thepleiotrophin is bFGF.
 43. The method of claim 41 wherein thepleiotrophin is CNTF.
 44. The method of claim 34 wherein the at leastone naturally occurring genetically encoded molecule that stimulatesneuritogenesis is a member of the S100 family of EF hand calcium bindingproteins.
 45. The method of claim 44 wherein the member of the S100family of EF hand calcium binding proteins is selected from the groupconsisting of S100β, p11, p9Ka, and calcyclin.
 46. The method of claim45 wherein the member of the S100 superfamily of EF hand calcium bindingproteins is S100β.
 47. The method of claim 45 wherein the member of theS-100 family of EF hand calcium binding proteins is p11.
 48. The methodof claim 45 wherein the member of the S-100 family of EF hand calciumbinding proteins is p9Ka.
 49. The method of claim 45 wherein the memberof the S-100 family of EF hand calcium binding proteins is calcyclin.50. The method of claim 34 wherein the at least one naturally occurringgenetically encoded molecule that stimulates neuritogenesis is a memberof the TGFβ superfamily.
 51. The method of claim 50 wherein the memberof the TGFβ superfamily is selected from the group consisting of TGFβ₁and GDNF.
 52. The method of claim 51 wherein the member of the TGFβsuperfamily is TGFβ₁.
 53. The method of claim 51 wherein the member ofthe TGFβ₁ superfamily is GDNF.
 54. The method of claim 32 wherein theinducing of the in vivo genetic expression of at least one naturallyoccurring genetically encoded molecule occurs in astrocytes of themammal.
 55. The method of claim 32 wherein the inducing of the in vivogenetic expression of at least one naturally occurring geneticallyencoded molecule activates the mitogen-activated protein kinase cascade.56. The method of claim 32 wherein the carbon monoxide dependentguanylyl cyclase modulating purine derivative is selected from the groupconsisting of guanosine, inosine pranobex, and a compound of formula (I)where n is an integer from 1 to 6, or of a salt or prodrug ester of acompound of formula (I) where n is an integer from 1 to
 6. 57. Themethod of claim 56 wherein the compound is a compound of formula (I)wherein n is an integer from 1 to
 6. 58. The method of claim 57 whereinn is 2 and wherein the compound isN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl)propanamide.
 59. The method ofclaim 32 wherein the effective amount of the at least one carbonmonoxide dependent guanylyl cyclase modulating purine derivativeproduces a treating concentration of at least 1 μM.
 60. The method ofclaim 32 wherein the at least one carbon monoxide dependent guanylylcyclase modulating purine derivative is orally administered to themammal.
 61. The method of claim 32 wherein the at least one carbonmonoxide dependent guanylyl cyclase modulating purine derivative isadministered to the mammal by injection.
 62. The method of claim 32wherein the mammal is a human.
 63. A method for modifying the membranepotential of a mammalian neuron comprising the step of administering aneffective amount of at least one carbon monoxide dependent guanylylcyclase modulating purine derivative to the mammalian neuron.
 64. Themethod of claim 63 wherein the effective amount of the at least onecarbon monoxide dependent guanylyl cyclase is administered to a mammalso that the method produces an increased learning capability in themammal.
 65. The method of claim 63 wherein the carbon monoxide dependentguanylyl cyclase modulating purine derivative is selected from the groupconsisting of guanosine, inosine pranobex, and a compound of formula (I)where n is an integer from 1 to 6, or of a salt or prodrug ester of acompound of formula (I) where n is an integer from 1 to
 6. 66. Themethod of claim 65 wherein the compound is a compound of formula (I)wherein n is an integer from 1 to
 6. 67. The method of claim 66 whereinn is 2 and wherein the compound isN-4-carboxyphenyl-3-(6-oxohydropurin-9-yl)propanamide.
 68. A method forselectively and controllably inducing the in vivo genetic expression ofat least one naturally occurring genetically encoded molecule in amammal comprising the step of administering an effective amount of atleast one carbon monoxide dependent guanylyl cyclase modulating guaninederivative to the mammal, the guanine derivative comprising a guaninemoiety linked through its nitrogen-9 atom through a linker to aphysiologically active group.
 69. The method of claim 68 wherein thelinker of the guanine derivative incorporates a hydrocarbyl moiety thatincludes a carbonyl group at one end.
 70. The method of claim 69 whereinthe end of the hydrocarbyl moiety that is terminated with the carbonylgroup is linked to the physiologically active group through an amidelinkage.
 71. The method of claim 68 wherein the at least one naturallygenetically encoded molecule stimulates neuritogenesis.
 72. The methodof claim 71 wherein the at least one naturally occurring geneticallyencoded molecule that stimulates neuritogenesis is selected from thegroup consisting of neurotrophins, pleiotrophins, members of the S100family of EF hand calcium binding proteins, and members of the TGFβsuperfamily.
 73. The method of claim 68 wherein the guanine derivativecomprises a compound of formula (II)

wherein n is an integer from 1 to
 6. 74. The method of claim 73 whereinn is 2 and the compound isN-4-carboxyphenyl-3-(2-amino-6-oxohydropurin-9-yl)propanamide.
 75. Themethod of claim 68 wherein the inducing of the in vivo geneticexpression of at least one naturally occurring genetically encodedmolecule occurs in astrocytes of the mammal.
 76. The method of claim 68wherein the inducing of the in vivo genetic expression of at least onenaturally occurring genetically encoded molecule activates themitogen-activated protein kinase cascade.
 77. A method for theadministration of at least one naturally occurring genetically encodedmolecule to a mammal comprising the step of selectably inducing the invivo genetic expression of the molecule in the mammal through theadministration of an effective amount of at least one carbon monoxidedependent guanylyl cyclase modulating guanine derivative to the mammalto raise the concentration of the at least one naturally occurringgenetically encoded molecule in at least one tissue of the mammal andthus cause the administration of the at least one naturally occurringgenetically encoded molecule to the mammal, the guanine derivativecomprising a guanine moiety linked through its nitrogen-9 atom through alinker to a physiologically active group.
 78. The method of claim 77wherein the linker of the guanine derivative incorporates a hydrocarbylmoiety that includes a carbonyl group at one end.
 79. The method ofclaim 78 wherein the end of the hydrocarbyl moiety that is terminatedwith the carbonyl group is linked to the physiologically active groupthrough an amide linkage.
 80. The method of claim 77 wherein the atleast one naturally genetically encoded molecule stimulatesneuritogenesis.
 81. The method of claim 80 wherein the at least onenaturally occurring genetically encoded molecule that stimulatesneuritogenesis is selected from the group consisting of neurotrophins,pleiotrophins, members of the S100 family of EF hand calcium bindingproteins, and members of the TGFβ superfamily.
 82. The method of claim77 wherein the guanine derivative comprises a compound of formula (II)wherein n is an integer from 1 to
 6. 83. The method of claim 82 whereinn is 2 and the compound isN-4-carboxyphenyl-3-(2-amino-6-oxohydropurin-9-yl)propanamide.
 84. Themethod of claim 77 wherein the inducing of the in vivo geneticexpression of at least one naturally occurring genetically encodedmolecule occurs in astrocytes of the mammal.
 85. The method of claim 77wherein the inducing of the in vivo genetic expression of at least onenaturally occurring genetically encoded molecule activates themitogen-activated protein kinase cascade.